U.S. patent application number 10/032818 was filed with the patent office on 2003-05-15 for inhibitors of memapsin 2 and use thereof.
This patent application is currently assigned to Oklahoma Medical Research Foundation. Invention is credited to Ghosh, Arun K., Koelsch, Gerald, Tang, Jordan J. N..
Application Number | 20030092629 10/032818 |
Document ID | / |
Family ID | 26946817 |
Filed Date | 2003-05-15 |
United States Patent
Application |
20030092629 |
Kind Code |
A1 |
Tang, Jordan J. N. ; et
al. |
May 15, 2003 |
Inhibitors of memapsin 2 and use thereof
Abstract
Methods for the production of purified, catalytically active,
recombinant memapsin 2 have been developed-, The substrate and
subsite specificity of the catalytically active enzyme have been
determined by a method which determines the initial hydrolysis rate
of the substrates by using MALDI-TOF/MS. Alternatively, the subsite
specificity of memapsin can be determined by probing a library of
inhibitors with memapsin 2 and subsequently detecting the bound
memapsin 2 with an antibody raised to memapsin 2 and an alkaline
phosphatase conjugated secondary antibody. The substrate and
subsite specificity information was used to design substrate
analogs of the natural memapsin 2 substrate that can inhibit the
function of memapsin 2. The substrate analogs are based on peptide
sequences, shown to be related to the natural peptide substrates
for memapsin 2. The substrate analogs contain at least one analog
of an amide bond which is not capable of being cleaved by memapsin
2. Processes for the synthesis of substrate analogues including
isosteres at the sites of the critical amino acid residues were
developed and the more than seventy substrate analogues were
synthesized, among which MMI-005, MMI-012, MMI-017, MMI-018,
MMI-025, MMI-026, MMI-037, MMI-039, MMI-040, MMI-066, MMI-070, and
MMI-071 have inhibition constants in the range of
1.4-61.4.times.10.sup.-9 M against recombinant pro-memapsin 2.
These inhibitors are useful in diagnostics and for the treatment
and/or prevention of Alzheimer's disease.
Inventors: |
Tang, Jordan J. N.; (Edmond,
OK) ; Koelsch, Gerald; (Oklahoma City, OK) ;
Ghosh, Arun K.; (River Forest, IL) |
Correspondence
Address: |
HAMILTON, BROOK, SMITH & REYNOLDS, P.C.
530 VIRGINIA ROAD
P.O. BOX 9133
CONCORD
MA
01742-9133
US
|
Assignee: |
Oklahoma Medical Research
Foundation
Oklahoma City
OK
|
Family ID: |
26946817 |
Appl. No.: |
10/032818 |
Filed: |
December 28, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60275756 |
Mar 14, 2001 |
|
|
|
60258705 |
Dec 28, 2000 |
|
|
|
Current U.S.
Class: |
514/17.8 ;
514/20.1; 530/326 |
Current CPC
Class: |
C12N 9/6478 20130101;
C07K 14/8142 20130101; C07K 2299/00 20130101; A61P 43/00 20180101;
C07K 5/0207 20130101; A61P 25/28 20180101; A61K 38/00 20130101;
G01N 33/6896 20130101; C12Q 1/37 20130101 |
Class at
Publication: |
514/13 ;
530/326 |
International
Class: |
A61K 038/10; C07K
007/08 |
Claims
What is claimed is:
1. An inhibitor of catalytically active memapsin 2 which binds to
the active site of the memapsin 2 defined by the presence of two
catalytic aspartic residues and substrate binding cleft, the
inhibitor having an K.sub.i of less than or equal to 10.sup.-7
M.
2. The inhibitor of claim 1, comprising an isostere of the active
site of memapsin 2.
3. The inhibitor of claim 2, comprising a molecule having the
general form
X-L.sub.4-P.sub.4-L.sub.3-P.sub.3-L.sub.2-P.sub.2-L.sub.1-P.sub.1-L.sub.0-
-P.sub.1'-L.sub.1'-P.sub.2'-L.sub.2'-P.sub.3'-L.sub.3'-P.sub.4'-L.sub.4'-Y-
, wherein P.sub.x represents the substrate specificity position
relative to the cleavage site which is represented by an -L.sub.0-,
and L.sub.x represent the linking regions between each substrate
specificity position, P.sub.x and wherein L.sub.0 is a
non-hydrolyzable bond and P.sub.1' is --R.sub.1CR.sub.3--, wherein
R.sub.1 is a group smaller than CH.sub.2OH (side chain of serine),
and at least two other P positions are a hydrophobic group.
4. The inhibitor of claim 1 having a K.sub.i of less than or equal
to 10.sup.-6 M.
5. The inhibitor of claim 1 having a K.sub.i of less than or equal
to 2 nM.
6. The inhibitor of claim 1 having a K.sub.i of less than or equal
to 1 nM.
7. The inhibitor of claim 1 having a root mean square difference of
less than or equal to 0.5 .ANG. for the side chain and backbone
atoms for amino acids 28-441 of SEQ ID NO: 2.
8. The inhibitor of claim 1 which is permeable to the blood brain
barrier.
9. The inhibitor of claim 1 which is less than 900 daltons in
molecular weight.
10. The inhibitor of claim 1 which blocks cleavage by memapsin 2
under physiological conditions.
11. The inhibitor of claim 1 having a K.sub.i of less than or equal
to 10.sup.-6 M.
12. A compound selected from the group consisting of MMI-005,
MMI-012, MMI-017, MMI-018, MMI-025, MMI-026, MMI-037, MMI-039,
MMI-040, MMI-065, MMI-066, MMI-070, and MMI-071.
13. A compound selected from the group consisting of MMI-012,
MMI-017, MMI-018, MMI-026, MMI-037, MMI-039, MMI-040, MMI-070 and
MMI-071.
14. A method for treating a patient to decrease the likelihood of
developing or the progression of Alzheimer's disease comprising
administering to the individual an effective amount of an inhibitor
of memapsin 2 selected from the group consisting of MMI-005,
MMI-012, MMI-017, MMI-018, MMI-025, MMI-026, MMI-037, MMI-039,
MMI-040, MMI-065, MMI-070 and MMI-071.
15. The method of claim 14, wherein the inhibitor is administered
orally.
16. The method of claim 14, wherein the inhibitor blocks cleavage
of APP.
17. A method of determining the substrate side-chain preference in
memapsin 2 sub-sites, comprising the steps of: a) reacting a
mixture of memapsin 2 substrates with memapsin 2; and b)
determining the sub-site preference of memapsin 2 by determining
relative initial hydrolysis rates of the mixture of memapsin 2
substrates.
18. The method of claim 17, wherein the initial hydrolysis rate is
determined by using a MALDI-TOF/MS device wherein the ion
intensities from the MALDI-TOF/MS measurements are used for
quantification of relative amounts of the substrates and the
products formed thereof by hydrolysis of the substrates.
19. A method of determining the substrate side-chain preference in
memapsin 2 sub-sites, comprising the steps of: a) preparing a
combinatorial library of memapsin 2 inhibitors wherein the
inhibitors comprise a base sequence taken from OM99-2; b) probing
the library of inhibitors with memapsin 2 wherein the memapsin 2
may bind one or a plurality of inhibitors to generate one or a
plurality of bound memapsin 2; and c) detecting the bound memapsin
2 with an antibody raised to memapsin 2 and an alkaline phosphatase
conjugated secondary antibody.
20. The method of claim 19, wherein the inhibitors are immobilized
on beads.
21. The method of claim 19, wherein the base sequence is EVNL*AAEF
and wherein the P.sub.3, P.sub.2, P.sub.2', and P.sub.3' sub-sites
of memapsin 2 are randomized.
22. A method of treating a human suffering from Alzheimer's disease
comprising administering to the human an inhibitor of catalytically
active memapsin 2 which binds to the active site of the memapsin 2
defined by the presence of two catalytic aspartic residues and
substrate binding cleft, the inhibitor having an K.sub.i of less
than or equal to 10.sup.-7 M.
23. A method of treating a human suffering from Alzheimer's disease
comprising administering to the human an inhibitor of catalytically
active memapsin 2 which binds to the active site of the memapsin 2
defined by the presence of two catalytic aspartic residues and
substrate binding cleft, the inhibitor having an K.sub.i of less
than or equal to 10.sup.-7 M, wherein the inhibitor has a root mean
square difference of less than or equal to 0.5 .ANG. for the side
chain and backbone atoms for amino acids 28-441 of SEQ ID NO:
2.
24. A method of treating a human suffering from Alzheimer's disease
comprising administering to the human a compound selected from the
group consisting of MMI-012, MMI-017, MMI-018, MMI-026, MMI-037,
MMI-039, MMI-040, MMI-070 and MMI-071.
Description
RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/275,756, filed on Mar. 14, 2001 and 60/258,705
filed on Dec. 28, 2000, the entire teachings of both of which are
incorporated by reference in their entirety.
BACKGROUND OF THE INVENTION
[0002] This invention is in the area of the design and synthesis of
specific inhibitors of the aspartic protease Memapsin 2
(beta-secretase, or .beta.-secretase) which are useful in the
treatment and/or prevention of Alzheimer's Disease.
[0003] Alzheimer's disease (AD) is a degenerative disorder of the
brain first described by Alios Alzheimer in 1907 after examining
one of his patients who suffered drastic reduction in cognitive
abilities and had generalized dementia (The early story of
Alzheimer's Disease, edited by Bick et al. (Raven Press, New York
1987)). It is the leading cause of dementia in elderly persons. AD
patients have increased problems with memory loss and intellectual
functions which progress to the point where they cannot function as
normal individuals. With the loss of intellectual skills the
patients exhibit personality changes, socially inappropriate
actions and schizophrenia (A Guide to the Understanding of
Alzheimer's Disease and Related Disorders, edited by Jorm (New York
University Press, New York 1987)). AD is devastating for both
victims and their families, for there is no effective palliative or
preventive treatment for the inevitable neurodegeneration.
[0004] AD is associated with neuritic plaques measuring up to 200
.mu.m in diameter in the cortex, hippocampus, subiculum,
hippocampal gyrus, and amygdala. One of the principal constituents
of neuritic plaques is amyloid, which is stained by Congo Red
(Fisher (1983); Kelly Microbiol. Sci. 1(9):214-219 (1984)). Amyloid
plaques stained by Congo Red are extracellular, pink or
rust-colored in bright field, and birefringent in polarized light.
The plaques are composed of polypeptide fibrils and are often
present around blood vessels, reducing blood supply to various
neurons in the brain.
[0005] Various factors such as genetic predisposition, infectious
agents, toxins, metals, and head trauma have all been suggested as
possible mechanisms of AD neuropathy. Available evidence strongly
indicates that there are distinct types of genetic predispositions
for AD. First, molecular analysis has provided evidence for
mutations in the amyloid precursor protein (APP) gene in certain
AD-stricken families (Goate et al. Nature 349:704-706 (1991);
Murrell et al. Science 254:97-99 (1991); Chartier-Harlin et al.
Nature 353:844-846 (1991); Mullan et al., Nature Genet. 1:345-347
(1992)). Additional genes for dominant forms of early onset AD
reside on chromosome 14 and chromosome 1 (Rogaev et al., Nature
376:775-778 (1995); Levy-Lahad et al., Science 269:973-977 (1995);
Sherrington et al., Nature 375:754-760 (1995)). Another loci
associated with AD resides on chromosome 19 and encodes a variant
form of apolipoprotein E (Corder, Science 261:921-923 (1993)).
[0006] Amyloid plaques are abundantly present in AD patients and in
Down's Syndrome individuals surviving to the age of 40. The
overexpression of APP in Down's Syndrome is recognized as a
possible cause of the development of AD in Down's patients over
thirty years of age (Rumble et al., New England J. Med.
320:1446-1452 (1989); Mann et al., Neurobiol. Aging 10:397-399
(1989)). The plaques are also present in the normal aging brain,
although at a lower number. These plaques are made up primarily of
the amyloid .beta. peptide (A.beta.; sometimes also referred to in
the literature as .beta.-amyloid peptide or .beta. peptide)
(Glenner and Wong, Biochem. Biophys. Res. Comm. 120:885-890
(1984)), which is also the primary protein constituent in
cerebrovascular amyloid deposits. The amyloid is a filamentous
material that is arranged in beta-pleated sheets. A.beta. is a
hydrophobic peptide comprising up to 43 amino acids.
[0007] The determination of its amino acid sequence led to the
cloning of the APP cDNA (Kang et al., Nature 325:733-735 (1987);
Goldgaber et al., Science 235:877-880 (1987); Robakis et al., Proc.
Natl. Acad. Sci. 84:4190-4194 (1987); Tanzi et al., Nature
331:528-530 (1988)) and genomic APP DNA (Lemaire et al., Nucl.
Acids Res. 17:517-522 (1989); Yoshikai et al., Gene 87, 257-263
(1990)). A number of forms of APP cDNA have been identified,
including the three most abundant forms, APP695, APP75 1, and
APP770. These forms arise from a single precursor RNA by alternate
splicing. The gene spans more than 175 kb with 18 exons (Yoshikai
et al. (1990)). APP contains an extracellular domain, a
transmembrane region and a cytoplasmic domain. A.beta. consists of
up to 28 amino acids just outside the hydrophobic transmembrane
domain and up to 15 residues of this transmembrane domain. A.beta.
is normally found in brain and other tissues such as heart, kidney
and spleen. However, A.beta. deposits are usually found in
abundance only in the brain.
[0008] Van Broeckhaven et al., Science 248:1120-1122 (1990), have
demonstrated that the APP gene is tightly linked to hereditary
cerebral hemorrhage with amyloidosis (HCHWA-D) in two Dutch
families. This was confirmed by the finding of a point mutation in
the APP coding region in two Dutch patients (Levy et al., Science
248:1124-1128 (1990)). The mutation substituted a glutamine for
glutamic acid at position 22 of the A.beta. (position 618 of
APP695, or position 693 of APP770). In addition, certain families
are genetically predisposed to Alzheimer's disease, a condition
referred to as familial Alzheimer's disease (FAD), through
mutations resulting in an amino acid replacement at position 717 of
the full length protein (Goate et al. (1991); Murrell et al.
(1991); Chartier-Harlin et al. (1991)). These mutations
co-segregate with the disease within the families and are absent in
families with late-onset AD. This mutation at amino acid 717
increases the production of the A.beta..sub.1-42 form of A.beta.
from APP (Suzuki et al., Science 264:1336-1340 (1994)). Another
mutant form contains a change in amino acids at positions 670 and
671 of the full length protein (Mullan et al. (1992)). This
mutation to amino acids 670 and 671 increases the production of
total A.beta. from APP (Citron et al., Nature 360:622-674
(1992)).
[0009] APP is processed in vivo at three sites. The evidence
suggests that cleavage at the .beta.-secretase site by a membrane
associated metalloprotease is a physiological event. This site is
located in APP 12 residues away from the lumenal surface of the
plasma membrane. Cleavage of the .beta.-secretase site (28 residues
from the plasma membrane's lumenal surface) and the
.beta.-secretase site (in the transmembrane region) results in the
40/42-residue .beta.-amyloid peptide (A .beta.), whose elevated
production and accumulation in the brain are the central events in
the pathogenesis of Alzheimer's disease (for review, see Selkoe, D.
J. Nature 399:23-31 (1999)). Presenilin 1, another membrane protein
found in human brain, controls the hydrolysis at the APP .gamma.
.beta.-secretase site and has been postulated to be itself the
responsible protease (Wolfe, M. S. et al., Nature 398:513-517
(1999)). Presenilin 1 is expressed as a single chain molecule and
its processing by a protease, presenilinase, is required to prevent
it from rapid degradation (Thinakaran, G. et al., Neuron 17:181-190
(1996) and Podlisny, M. B., et al., Neurobiol. Dis. 3:325-37
(1997)). The identity of presenilinase is unknown. The in vivo
processing of the .beta.-secretase site is thought to be the
rate-limiting step in A.beta. production (Sinha, S. &
Lieberburg, I., Proc. Natl. Acad. Sci., USA, 96:11049-11053
(1999)), and is therefore a strong therapeutic target.
[0010] The design of inhibitors effective in decreasing amyloid
plaque formation is dependent on the identification of the critical
enzyme(s) in the cleavage of APP to yield the 42 amino acid
peptide, the A.beta..sub.1-42 form of A.beta.. Although several
enzymes have been identified, it has not been possible to produce
active enzyme. Without active enzyme, one cannot confirm the
substrate specificity, determine the subsite specificity, nor
determine the kinetics or critical active site residues, all of
which are essential for the design of inhibitors.
[0011] Memapsin 2 has been shown to be beta-secretase, a key
protease involved in the production in human brain of beta-amyloid
peptide from beta-amyloid precursor protein (for review, see
Selkoe, D. J. Nature 399:23-31 (1999)). It is now generally
accepted that the accumulation of beta-amyloid peptide in human
brain is a major cause for Alzheimer's disease. Inhibitors
specifically designed for human memapsin 2 should inhibit or
decrease the formation of beta-amyloid peptide and the progression
of the Alzheimer's disease.
[0012] Memapsin 2 belongs to the aspartic protease family. It is
homologous in amino acid sequence to other eukaryotic aspartic
proteases and contains motifs specific to that family. These
structural similarities predict that memapsin 2 and other
eukaryotic aspartic proteases share common catalytic mechanism,
Davies, D. R., Annu. Rev. Biophys. Chem. 19, 189 (1990). The most
successful inhibitors for aspartic proteases are mimics of the
transition state of these enzymes. These inhibitors have
substrate-like structure with the cleaved planar peptide bond
between the carbonyl carbon and the amide nitrogen replaced by two
tetrahedral atoms, such as hydroxyethylene [--CH(OH)--CH.sub.2--],
which was originally discovered in the structure of pepstatin
(Marciniszyn et al., 1976).
[0013] A need exists to develop new, improved inhibitors of
proteases involved in the production of beta-amyloid protein from
beta-amyloid precursor protein, such as memapsin 2 inhibitors, that
are effective, for example, in the treatment of Alzheimer's disease
in humans.
SUMMARY OF THE INVENTION
[0014] The present invention relates to inhibitors of memapsin 2
activity and methods of using the inhibitors of memapsin 2 to treat
Alzheimer's disease in humans.
[0015] In one embodiment, the invention is an inhibitor of
catalytically active memapsin 2 which binds to the active site of
the memapsin 2 defined by the presence of two catalytic aspartic
residues and substrate binding cleft, the inhibitor having an K, of
less than or equal to 10.sup.-7 M.
[0016] In another embodiment, the invention includes a compound
selected from the group consisting of MMI-005, MMI-012, MMI-017,
MMI-018, MMI-025, MMI-026, MMI-037, MMI-039, MMI-040, MMI-065,
MMI-066, MMI-070, and MMI-071.
[0017] In yet another embodiment, the invention includes a compound
selected from the group consisting of MMI-012, MMI-017, MMI-018,
MMI-026, MMI-037, MMI-039, MMI-040, MMI-070 and MMI-071.
[0018] Another embodiment includes a method for treating a patient
to decrease the likelihood of developing or the progression of
Alzheimer's disease comprising administering to the individual an
effective amount of an inhibitor of memapsin 2 selected from the
group consisting of MMI-005, MMI-012, MMI-017, MMI-018, MMI-025,
MMI-026, MMI-037, MMI-039, MMI-040, MMI-065, MMI-070 and
MMI-071.
[0019] In an additional embodiment, the invention is a method of
determining the substrate side-chain preference in memapsin 2
sub-sites, comprising the steps of reacting a mixture of memapsin 2
substrates with memapsin 2 and determining the sub-site preference
of memapsin 2 by determining relative initial hydrolysis rates of
the mixture of memapsin 2 substrates.
[0020] In still another embodiment, the invention includes a method
of determining the substrate side-chain preference in memapsin 2
sub-sites. A combinatorial library of memapsin 2 inhibitors wherein
the inhibitors comprise a base sequence taken from OM99-2 is
prepared. The library of inhibitors is probed with memapsin 2
wherein the memapsin 2 may bind one or a plurality of inhibitors to
generate one or a plurality of bound memapsin 2 and the bound
memapsin 2 is detected with an antibody raised to memapsin 2 and an
alkaline phosphatase conjugated secondary antibody.
[0021] In a further embodiment, the invention includes a method of
treating a human suffering from Alzheimer's disease comprising
administering to the human an inhibitor of catalytically active
memapsin 2 which binds to the active site of the memapsin 2 defined
by the presence of two catalytic aspartic residues and substrate
binding cleft, the inhibitor having an K.sub.i of less than or
equal to 10.sup.-7 M.
[0022] In yet another embodiment, the invention relates to a method
of treating a human suffering from Alzheimer's disease comprising
administering to the human an inhibitor of catalytically active
memapsin 2 which binds to the active site of the memapsin 2 defined
by the presence of two catalytic aspartic residues and substrate
binding cleft, the inhibitor having an K.sub.i of less than or
equal to 10.sup.-7 M, wherein the inhibitor has a root mean square
difference of less than or equal to 0.5 .ANG. for the side chain
and backbone atoms for amino acids 28-441 of SEQ ID NO: 2.
[0023] An additional embodiment of the invention is a method of
treating a human suffering from Alzheimer's disease comprising
administering to the human a compound selected from the group
consisting of MMI-012, MMI-017, MMI-018, MMI-026, MMI-037, MMI-039,
MMI-040, MMI-070 and MMI-071.
[0024] In yet another embodiment, the invention relates to the use
of an inhibitor of catalytically active memapsin 2 which binds to
the active site of the memapsin 2 defined by the presence of two
catalytic aspartic residues and substrate binding cleft, the
inhibitor having an K.sub.i of less than or equal to 10.sup.-7 M
for the manufacture of a medicament for the treatment of
Alzheimer's disease in a human.
[0025] In an additional embodiment, the invention relates to the
use of an inhibitor of catalytically active memapsin 2 which binds
to the active site of the memapsin 2 defined by the presence of two
catalytic aspartic residues and substrate binding cleft, the
inhibitor having an K.sub.i of less than or equal to 10.sup.-7 M,
wherein the inhibitor has a root mean square difference of less
than or equal to 0.5 .ANG. for the side chain and backbone atoms
for amino acids 18-379 of memapsin 2, for the manufacture of a
medicament for the treatment of Alzheimer's disease in a human.
[0026] In still another embodiment, the invention relates to the
use of a compound selected from the group consisting of MMI-005,
MMI-012, MMI-017, MMJ-018, MMI-025, MMI-026, MMI-037, MMI-039,
MMI-040, MMI-065, MMI-066, MMI-070 and MMI-071, for the manufacture
of a medicament for the treatment of Alzheimer's disease in a
human.
[0027] The substrate and subsite specificity of recombinant,
catalytically active memapsin 2 was used to design substrate
analogs of the natural memapsin 2 substrate that can inhibit the
function of memapsin 2. Initially, X-ray crystallography of
memapsin 2 bound to a substrate analog, OM99-2, was used to
determine the three dimensional structure of the memapsin 2, as
well as the importance of the various residues in binding.
Substrate analogs were then designed based on peptide sequences
shown to be related to the natural peptide substrates for memapsin
2 and the crystallographic structure. The substrate analogs contain
at least one analog of an amide (peptide) bond that is not capable
of being cleaved by memapsin 2.
[0028] The substrate and subsite specificity of the catalytically
active memapsin 2 have been further determined by a method which
determines the initial hydrolysis rate of the substrates using mass
spectroscopy, for example, MALDI-TOF/MS (matrix assisted laser
desorption/ionization-time of flight/mass spectroscopy).
Alternatively, the subsite specificity of memapsin was further
determined by probing a library of inhibitors with memapsin 2 and
subsequently detecting the bound memapsin 2 with an antibody raised
to memapsin 2 and an alkaline phosphatase conjugated secondary
antibody. The substrate and subsite specificity information was
used to design additional substrate analogs of the natural memapsin
2 substrate that can inhibit the function of memapsin 2.
[0029] Processes for the synthesis of substrate analogues including
isosteres at the sites of the critical amino acid residues were
developed. Substrate analogues, OM99-1, OM99-2 and more than
seventy other substrate analogues were synthesized. OM99-2 is based
on an octapeptide Glu-Val-Asn-Leu-Ala-Ala-Glu-Phe (SEQ ID NO: 28)
with the Leu-Ala peptide bond substituted by a transition-state
isostere hydroxyethylene group. The inhibition constant of OM99-2
is 1.6.times.10.sup.-9 M against recombinant pro-memapsin 2. The
inhibition constants of MMI-005, MMI-012, MMI-017, MMI-018,
MMI-025, MMI-026, MMI-037, MMI-039, MMI-040, MMI-066, MMI-070, and
MMI-071 have inhibition constants in the range of
1.4-61.4.times.10.sup.-9 M against recombinant pro-memapsin 2.
[0030] Compositions that inhibit memapsin 2 aspartic protease
activity can be small molecules, which readily pass across the
blood brain barrier, are administered orally, and are not
inactivated by intestinal enzymes. Furthermore, it is desirable
that such compositions are relatively inexpensive to manufacture
and preferentially inhibit memapsin 2 cleavage of beta-amyloid
precursor protein.
[0031] This information can be used by those skilled in the art to
design additional new inhibitors, using commercially available
software programs and techniques familiar to those in organic
chemistry and enzymology, to design new inhibitors. For example,
the side chains of the inhibitors may be modified to produce
stronger interactions (through hydrogen bonding, hydrophobic
interaction, charge interaction and/or van der Waal interaction) in
order to increase inhibition potency. Based on this type of
information, the residues with minor interactions may be eliminated
from the new inhibitor design to decrease the molecular weight of
the inhibitor. The side chains with no structural hindrance from
the enzyme may be cross-linked to lock in the effective inhibitor
conformation. This type of structure also enables the design of
peptide surrogates which may effectively fill the binding sites of
memapsin 2 yet produce better pharmaceutical properties. The
rational design and screening of compounds for inhibitors, and
their characterization are provided in this invention. Compositions
effective for inhibition of memapsin 2 include small molecule
inhibitors, and inhibitors that are capable of crossing the blood
brain barrier. Such inhibitors can interact with memapsin 2, or its
substrate, to inhibit cleavage by memapsin 2.
[0032] The invention described herein provides compounds which
inhibit memapsin 2 activity, in particular the aspartic protease
activity of memapsin 2, which converts beta-amyloid precursor
protein to beta-amyloid protein. The compounds of the invention can
be used to treat Alzheimer's disease in humans.
BRIEF DESCRIPTION OF THE DRAWINGS
[0033] FIG. 1 is a schematic representation of inhibitors for
memapsin 2. "A" and "B" denote aliphatic linkages (saturated or
partially unsaturated) of any number of carbons, between side
chains in positions P.sub.1 and P.sub.3, for example, the amino
acid side chains P.sub.1 Leu and P.sub.3 Val in these respective
positions. Other structural elements of the inhibitor are omitted
for clarity.
[0034] FIG. 2 is a schematic representation of inhibitors for
memapsin 2. "A" denotes aliphatic linkages (saturated or partially
unsaturated) of any number of carbons, between side chains in
positions P.sub.2 and P.sub.4, for example, the amino acid side
chains P.sub.2 Asn and P.sub.4 Glu in these respective positions.
Other structural elements of the inhibitor are omitted for
clarity.
[0035] FIG. 3 is a schematic of the design for the side chain at
the P.sub.1' subsite for the new memapsin 2 inhibitors based on the
current crystal structure. Arrows indicate possible interactions
between memapsin 2 and inhibitor. Other structural elements of
inhibitor are omitted for clarity.
[0036] FIG. 4 is a schematic representation of inhibitors for
memapsin 2. "A" and "B" denote aliphatic linkages (saturated or
partially unsaturated) between side chains in positions P.sub.1 and
the backbone atoms of P.sub.3, for example, the amino acid side
chains P.sub.1 Leu in this position. Other structural elements of
the inhibitor are omitted for clarity.
[0037] FIG. 5 shows the relative specificity of memapsin 2 for
amino acid residues in positions P.sub.1'-P.sub.4'. Letters above
bars indicate the native amino acid at that position. Catalytic
efficiency is expressed relative to the native amino acid at each
position.
[0038] FIG. 6 depicts the nucleotide sequence of human Memapsin 2
(SEQ ID NO: 1).
[0039] FIGS. 7A and 7B depict the partial protein sequence of human
Memapsin 2, excluding the signal peptide (SEQ ID NO: 2). Amino
acids 28-48 are remnant putative propeptide residues. Amino acids
58-61, 78, 80, 82-83, 116, 118-121, 156, 166, 174, 246, 274, 276,
278-281, 283, and 376-377 are residues in contact with the OM99-2
inhibitor. Amino acids 54-57, 61-68, 73-80, 86-89, 109-111,
113-118, 123-134, 143-154, 165-168, 198-202, and 220-224 are N-lobe
beta strands. Amino acids 184-191 and 210-217 are N-lobe helices.
Amino acids 237-240, 247-249, 251-256, 259-260, 273-275, 282-285,
316-318, 331-336, 342-348, 354-357, 366-370, 372-375, 380-383,
390-395, 400-405, and 418-420 are C-lobe beta strands. Amino acids
286-299, 307-310, 350-353, 384-387, and 427-431 are C-lobe
helices.
[0040] FIGS. 8A and 8B depict the protein sequence of human
promemapsin 2 (SEQ ID NO: 3). Amino acids 1-15 are vector-derived
residues. Amino acids 16-63 are a putative pro peptide. Amino acids
1-13 are derived from the T7 promoter. Amino acids 16-456 are
Pro-memapsin 2-T1. Amino acids 16-421 are Promemapsin 2-T2.
[0041] FIG. 9 depicts the amino acid sequence of human
pre-promemapsin 2 (SEQ ID NO: 4). Amino acids 1-13 are the signal
peptide. Amino acids 41-454 correspond to amino acids 28-441 of
FIGS. 7A and 7B, and amino acids 43-456 of FIGS. 5A and 5B. The
active site aspartic acids are at amino acid positions 93 and
289.
[0042] The foregoing and other objects, features and advantages of
the invention will be apparent from the following more particular
description of preferred embodiments of the invention.
DETAILED DESCRIPTION OF THE INVENTION
[0043] The features and other details of the invention, either as
steps of the invention or as combinations of parts of the
invention, will now be more particularly described and pointed out
in the claims. It will be understood that the particular
embodiments of the invention are shown by way of illustration and
not as limitations of the invention. The principle features of this
invention can be employed in various embodiments without departing
from the scope of the invention.
[0044] I. Design and Synthesis of Inhibitors
[0045] Design of Substrate Analogs for Memapsin 2.
[0046] The five human aspartic proteases have homologous amino acid
sequences and have similar three-dimensional structures. There are
two aspartic residues in the active site and each residue is found
within the signature aspartic protease sequence motif,
Asp-Thr/Ser-Gly-. There are generally two homologous domains within
an aspartic protease and the substrate binding site is positioned
between these two domains, based on the three-dimensional
structures. The substrate binding sites of aspartic proteases
generally recognize eight amino acid residues. There are generally
four residues on each side of the amide bond that is cleaved by the
aspartic protease.
[0047] Typically the side chains of each amino acid are involved in
the specificity of the substrate/aspartic protease interaction. The
side chain of each substrate residue is recognized by regions of
the enzyme which are collectively called sub-sites. The generally
accepted nomenclature for the protease sub-sites and their
corresponding substrate residues are shown below, where the double
slash represents the position of bond cleavage.
1 Protease sub-sites S4 S3 S2 S1 S1' S2' S3' S4' Substrate residues
P4 P3 P2 P1 // P1' P2' P3' P4'
[0048] While there is a general motif for aspartic protease
substrate recognition, each protease has a very different substrate
specificity and breadth of specificity. Once the specificity of an
aspartic protease is known, inhibitors can be designed based on
that specificity, which interact with the aspartic protease in a
way that prevents natural substrate from being efficiently cleaved.
Some aspartic proteases have specificities that can accommodate
many different residues in each of the sub-sites for successful
hydrolysis. Pepsin and cathepsin D have this type of specificity
and are said to have "broad" substrate specificity. When only a
very few residues can be recognized at a sub-site, such as in
renin, the aspartic protease is said to have a stringent or narrow
specificity.
[0049] The information on the specificity of an aspartic protease
can be used to design specific inhibitors in which the preferred
residues are placed at specific sub-sites and the cleaved peptide
bond is replaced by an analog of the transition-state. These
analogs are called transition state isosteres. Aspartic proteases
cleave amide bonds by a hydrolytic mechanism. This reaction
mechanism involves the attack by a hydroxide ion on the
.beta.-carbon of the amino acid. Protonation must occur at the
other atom attached to the .beta.-carbon through the bond that is
to be cleaved. If the .beta.-carbon is insufficiently electrophilic
or the atom attached to the bond to be cleaved is insufficiently
nucleophilic, the bond will not be cleaved by a hydrolytic
mechanism. Analogs exist which do not mimic the transition state
but which are non-hydrolyzable, but transition state isosteres
mimic the transition state specifically and are
non-hydrolyzable.
[0050] Transition state theory indicates that it is the transition
state intermediate of the reaction which the enzyme catalyzes for
which the enzyme has its highest affinity. It is the transition
state structure, not the ground state structure, of the substrate
which will have the highest affinity for its given enzyme. The
transition state for the hydrolysis of an amide bond is tetrahedral
while the ground state structure is planar. A typical
transition-state isostere of aspartic protease is
--CH(OH)--CH.sub.2--, as was first discovered in pepstatin by
Marciniszyn et al. (1976). The transition-state analogue principles
have been successfully applied to inhibitor drugs for human
immunodeficiency virus protease, an aspartic protease. Many of
these are currently in clinical use. Information on the structure,
specificity, and types of inhibitors can be found in Tang, Acid
Proteases, Structure, Function and Biology, Adv. in Exptl. Med.
Biol. vol. 95 (Plenum Press, NY 1977); Kostka, Aspartic Proteinases
and their Inhibitors (Walter de Gruyter, Berlin 1985); Dunn,
Structure and Functions of the Aspartic Proteinases, Adv. in Exptl.
Med. Biol. 306 (Plenum Press, NY 1991); Takahashi, Aspartic
Proteases, Structure, Function, Biology, Biomedical Implications,
Adv. in Exptl. Med. Biol. 362 (Plenum Press, NY 1995); and James,
Aspartic Proteinases, Retroviral and Cellular Enzymes, Adv. in
Exptl. Med. Biol. 436 (Plenum Press, NY 1998), the teachings of all
of which are incorporated herein in their entirety).
[0051] Substrate analog compositions are generally of the general
formula
X-L.sub.4-P.sub.4-L.sub.3-P.sub.3-L.sub.2-P.sub.2-L.sub.1-P.sub.1-L.sub.0-
-P.sub.1'-L.sub.1'-P.sub.2'-L.sub.2'-P.sub.3'-L.sub.3'-P.sub.4'-L.sub.4'-Y-
. The substrate analog compositions are analogs of small peptide
molecules. Their basic structure is derived from peptide sequences
that were determined through structure/function studies. It is
understood that positions represented by P.sub.x represent the
substrate specificity position relative to the cleavage site which
is represented by an -L.sub.0-. The positions of the compositions
represented by L.sub.x represent the linking regions between each
substrate specificity position, P.sub.x.
[0052] In a natural substrate for memapsin 2, a P.sub.x-L.sub.x
pair would represent a single amino acid of the peptide which is to
be cleaved. In the present general formula, each P.sub.x part of
the formula refers to the .alpha.-carbon and side chain functional
group of each would be amino acid. Thus, the P.sub.x portion of an
P.sub.xL.sub.x pair for alanine represents HC--CH.sub.3. The
general formula representing the P.sub.x portion of the general
composition is --R.sub.1CR.sub.3--.
[0053] In general R.sub.1 can be either CH.sub.3 (side chain of
alanine), CH(CH.sub.3).sub.2 (side chain of valine),
CH.sub.2CH(CH.sub.3).sub.2 (side chain of leucine),
(CH.sub.3)CH(CH.sub.2CH.sub.3) (side chain of isoleucine),
CH.sub.2(indole) (side chain of tryptophan), CH.sub.2(benzene)
(side chain of phenylalanine), CH.sub.2CH.sub.2SCH.sub.- 3 (side
chain of methionine), H (side chain of glycine), CH.sub.2OH (side
chain of serine), CHOHCH.sub.3 (side chain of threonine),
CH.sub.2(phenol) (side chain of tyrosine), CH.sub.2SH (side chain
of cysteine), CH.sub.2CH.sub.2CONH.sub.2 (side chain of glutamine),
CH.sub.2CONH.sub.2 (side chain of asparagine),
CH.sub.2CH.sub.2CH.sub.2CH- .sub.2NH.sub.2 (side chain of lysine),
CH.sub.2CH.sub.2CH.sub.2NHC(NH)(NH.- sub.2) (side chain of
arginine), CH.sub.2(imidazole) (side chain of histidine),
CH.sub.2COOH (side chain of aspartic acid), CH.sub.2CH.sub.2COOH
(side chain of glutamic acid), and functional natural and
non-natural derivatives or synthetic substitutions of these.
[0054] It is most preferred that R.sub.3 is a single H. In general,
however, R.sub.3 can be alkenyl, alkynal, alkenyloxy, and
alkynyloxy groups that allow binding to memapsin 2. Preferably,
alkenyl, alkynyl, alkenyloxy and alkynyloxy groups have from 2 to
40 carbons, and more preferably from 2 to 20 carbons, from 2 to 10
carbons, or from 2 to 3 carbons, and functional natural and
non-natural derivatives or synthetic substitutions of these.
[0055] The L.sub.x portion of the P.sub.x-L.sub.x pair represents
the atoms linking the P.sub.x regions together. In a natural
substrate the L.sub.x represents the .beta.-carbon attached to the
amino portion of what would be the next amino acid in the chain.
Thus, L.sub.x would be represented by --CO--NH--. The general
formula for L.sub.x is represented by R.sub.2. In general R.sub.2
can be CO--HN (amide), CH(OH)(CH.sub.2) (hydroxyethylene),
CH(OH)CH(OH) (dihydroxyethylene), CH(OH)CH.sub.2NH
(hydroxyethylamine), PO(OH)CH.sub.2 (phosphinate), CH.sub.2NH
(reduced amide). It is understood that more than one L-- maybe an
isostere as long as the substrate analog functions to inhibit
aspartic protease function.
[0056] Ls which are not isosteres may either be an amide bond or
mimetic of an amide bond that is non-hydrolyzable.
[0057] X and Y represent molecules which are not typically involved
in the recognition by the aspartic protease recognition site, but
which do not interfere with recognition. It is preferred that these
molecules confer resistance to the degradation of the substrate
analog. Preferred examples would be amino acids coupled to the
substrate analog through a non-hydrolyzable bond. Other preferred
compounds are capping agents. Still other preferred compounds are
compounds that could be used in the purification of the substrate
analogs such as biotin.
[0058] As used herein, alkyl refers to substituted or unsubstituted
straight, branched or cyclic alkyl groups; and alkoxyl refers to
substituted or unsubstituted straight, branched or cyclic alkoxy.
Preferably, alkyl and alkoxy groups have from 1 to 40 carbons, and
more preferably from 1 to 20 carbons, from 1 to 10 carbons, or from
1 to 3 carbons.
[0059] As used herein, alkenyl refers to substituted or
unsubstituted straight chain or branched alkenyl groups; alkynyl
refers to substituted or unsubstituted straight chain or branched
alkynyl groups; alkenyloxy refers to substituted or unsubstituted
straight chain or branched alkenyloxy; and alkynyloxy refers to
substituted or unsubstituted straight chain or branched alkynyloxy.
Preferably, alkenyl, alkynyl, alkenyloxy and alkynyloxy groups have
from 2 to 40 carbons, and more preferably from 2 to 20 carbons,
from 2 to 10 carbons, or from 2 to 3 carbons.
[0060] As used herein, alkaryl refers to an alkyl group that has an
aryl substituent; aralkyl refers to an aryl group that has an alkyl
substituent; heterocyclic-alkyl refers to a heterocyclic group with
an alkyl substituent; alkyl-heterocyclic refers to an alkyl group
that has a heterocyclic substituent.
[0061] The substituents for alkyl, alkenyl, alkynyl, alkoxy,
alkenyloxy, and alkynyloxy groups can be halogen, cyano, amino,
thio, carboxy, ester, ether, thioether, carboxamide, hydroxy, or
mercapto. Further, the groups can optionally have one or more
methylene groups replaced with a heteroatom, such as O, NH or
S.
[0062] A number of different substrates were tested and analyzed,
and the cleavage rules for Memapsin 2 were determined. The results
of the substrates which were analyzed are presented in Table 1 and
the rules determined from these results are summarized below.
[0063] (1) The primary specificity site for a memapsin 2 substrate
is subsite position, S.sub.1'. This means that the most important
determinant for substrate specificity in memapsin 2 is the amino
acid, P1'. P.sub.1' may be a small side chain for memapsin 2 to
recognize the substrate. Preferred embodiments are substrate
analogs where R.sub.1 of the P.sub.1' position is either H (side
chain of glycine), CH.sub.3 (side chain of alanine), CH.sub.2OH
(side chain of serine), or CH.sub.2COOH (side chain of aspartic
acid). Embodiments that have an R.sub.1 structurally smaller than
CH.sub.3 (side chain of alanine) or CH.sub.2OH (side chain of
serine) are also preferred. However, substrates in Table 1 nay not,
due to their amino acid composition, provide a complete
representation of the substrate specificity of memapsin 2.
Therefore, P.sub.1' may not be limited to the small residues
mentioned, but may also include the following:
[0064] CH.sub.2CH(CH.sub.3).sub.2 (sidechain of leucine),
[0065] (CH.sub.3)CH(CH.sub.2CH.sub.3) (sidechain of
isoleucine),
[0066] CH.sub.2(INDOLE) (sidechain of tryptophan),
[0067] CH.sub.2(BENZNE) (sidechain of phenylalanine),
[0068] CH(CH.sub.3).sub.2 (sidechain of valine),
[0069] CH.sub.2(PHENOL) (sidechain of tyrosine),
[0070] CH.sub.2CH.sub.2SCH.sub.3 (sidechain of methionine),
[0071] CH(CH.sub.3OH)(sidechain of threonine),
[0072] CH.sub.2CONH (sidechain of asparagine),
[0073] CH.sub.2CH.sub.2CONH (sidechain of glutamine),
[0074] CH.sub.2CH.sub.2COOH (sidechain of glutamic acid),
[0075] CH.sub.2CH.sub.2CH.sub.2CH.sub.2NH.sub.3(sidechain of
lysine),
[0076] CH.sub.2CH.sub.2CH.sub.2NC(NH.sub.2).sub.2 (sidechain of
arginine),
[0077] CH.sub.2(IMIDAZOLE) (sidechain of histidine).
[0078] (2) There are no specific sequence requirements at positions
P.sub.4 P.sub.3, P.sub.2, P.sub.1, P.sub.2', P.sub.3', and
P.sub.4'. Each site can accommodate any other amino acid residue in
singularity as long as rule number 3 is met.
[0079] (3) At least two of the remaining seven positions, P.sub.4,
P.sub.3, P.sub.2, P.sub.1, P.sub.2', P.sub.3', and P.sub.4', must
have an R.sub.1 which is made up of a hydrophobic residue. It is
preferred that there are at least three hydrophobic residues in the
remaining seven positions, P.sub.4, P.sub.3, P.sub.2, P.sub.1,
P.sub.2', P.sub.3', and P.sub.4'. Preferred R.sub.1 groups for the
positions that contain a hydrophobic group are CH.sub.3 (side chain
of alanine), CH(CH.sub.3).sub.2 (side chain of valine),
CH.sub.2CH(CH.sub.3).sub.2 (side chain of leucine),
(CH.sub.3)CH(CH.sub.2 CH.sub.3) (side chain of isoleucine),
CH.sub.2(indole) (side chain of tryptophan), CH.sub.2(benzene)
(side chain of phenylalanine), CH.sub.2CH.sub.2SCH.sub.- 3 (side
chain of methionine) CH.sub.2(phenol) (side chain of tyrosine). It
is more preferred that the hydrophobic group be a large hydrophobic
group. Preferred R.sub.1s which contain large hydrophobic groups
are CH(CH.sub.3).sub.2 (side chain of valine),
CH.sub.2CH(CH.sub.3).sub.2 (side chain of leucine),
(CH.sub.3)CH(CH.sub.2 CH.sub.3) (side chain of isoleucine),
CH.sub.2(indole) (side chain of tryptophan), CH.sub.2(benzene)
(side chain of phenylalanine), CH.sub.2CH.sub.2SCH.sub.- 3 (side
chain of methionine) CH.sub.2(phenol) (side chain of tyrosine). It
is most preferred that positions with a hydrophobic R.sub.1 are
CH(CH.sub.3).sub.2 (side chain of valine),
CH.sub.2CH(CH.sub.3).sub.2 (side chain of leucine),
CH.sub.2(benzene) (side chain of phenylalanine),
CH.sub.2CH.sub.2SCH.sub.3 (side chain of methionine), or
CH.sub.2(phenol) (side chain of tyrosine).
[0080] (4) None of the eight positions, P.sub.4, P.sub.3, P.sub.2,
P.sub.1, P.sub.1,' P.sub.2', P.sub.3', and P.sub.4' may have a
proline side chain at its R1 position.
[0081] (5) Not all subsites must have an P represented in the
analog. For example, a substrate analog could have
X-P.sub.2-L.sub.1-P.sub.1-L.sub.0--
P.sub.1'-L.sub.1'-P.sub.2'-L.sub.2'-P.sub.3'-L.sub.3'-Y or it could
have
X-L.sub.1-P.sub.1-L.sub.0-P.sub.1'-L.sub.1'-P.sub.2'-L.sub.2'-P.sub.3'-L.-
sub.3'-P.sub.4'-L.sub.4'-Y.
[0082] Preferred substrate analogs are analogs having the sequences
disclosed in Table 1, with the non-hydrolyzable analog between P1
and P1'.
[0083] Combinatorial Chemistry to Make Inhibitors
[0084] Combinatorial chemistry includes but is not limited to all
methods for isolating molecules that are capable of binding either
a small molecule or another macromolecule. Proteins,
oligonucleotides, and polysaccharides are examples of
macromolecules. For example, oligonucleotide molecules with a given
function, catalytic or ligand-binding, can be isolated from a
complex mixture of random oligonucleotides in what has been
referred to as "in vitro genetics" (Szostak, TIBS 19:89, 1992, the
teachings of which are incorporated herein in their entirety). One
synthesizes a large pool of molecules bearing random and defined
sequences and subjects that complex mixture, for example,
approximately 10.sup.15 individual sequences in 100 .mu.g of a 100
nucleotide RNA, to some selection and enrichment process. Through
repeated cycles of affinity chromatography and PCR amplification of
the molecules bound to the ligand on the column, Ellington and
Szostak (1990) estimated that 1 in 10.sup.10 RNA molecules folded
in such a way as to bind a small molecule dyes. DNA molecules with
such ligand-binding behavior have been isolated as well (Ellington
and Szostak, 1992; Bock et al, 1992, the teachings of all of which
are incorporated herein in their entirety).
[0085] Techniques aimed at similar goals exist for small organic
molecules, proteins and peptides and other molecules known to those
of skill in the art. Screening sets of molecules for a desired
activity whether based on libraries of small synthetic molecules,
oligonucleotides, proteins or peptides is broadly referred to as
combinatorial chemistry.
[0086] There are a number of methods for isolating proteins either
have de novo activity or a modifed activity. For example, phage
display libraries have been used for a number of years. A preferred
method for isolating proteins that have a given function is
described by Roberts and Szostak (Roberts R. W. and Szostak J. W.
Proc. Natl. Acad. Sci. USA, 94 (23)12997-302 (1997), the teachings
of which are incorporated herein in their entirety). Another
preferred method for combinatorial methods designed to isolate
peptides is described in Cohen et al. (Cohen B. A., et al., Proc.
Natl. Acad. Sci. USA 95 (24):14272-7 (1998), the teachings of which
are incorporated herein in their entirety). This method utilizes a
modified two-hybrid technology. Yeast two-hybrid systems are useful
for the detection and analysis of protein:protein interactions. The
two-hybrid system, initially described in the yeast Saccharomyces
cerevisiae, is a powerful molecular genetic technique for
identifying new regulatory molecules, specific to the protein of
interest (Fields and Song, Nature 340:245-6 (1989), the teachings
of which are incorporated herein in their entirety). Cohen et al.,
modified this technology so that novel interactions between
synthetic or engineered peptide sequences could be identified which
bind a molecule of choice. The benefit of this type of technology
is that the selection is done in an intracellular environment. The
method utilizes a library of peptide molecules that attach to an
acidic activation domain. A peptide of choice, for example an
extracellular portion of memapsin 2 is attached to a DNA binding
domain of a transcriptional activation protein, such as Gal 4. By
performing the Two-hybrid technique on this type of system,
molecules that bind the extracellular portion of memapsin 2 can be
identified.
[0087] Screening of Small Molecule Libraries
[0088] In addition to these more specialized techniques,
methodology well known to those of skill in the art, in combination
with various small molecule or combinatorial libraries, can be used
to isolate and characterize those molecules which bind to or
interact with the desired target, either memapsin 2 or its
substrate. The relative binding affinity of these compounds can be
compared and optimum inhibitors identified using competitive or
non-competitive binding studies which are well known to those of
skill in the art. Preferred competitive inhibitors are
non-hydrolyzable analogs of memapsin 2. Another will cause
allosteric rearrangements which prevent memapsin 2 from functioning
or folding correctly.
[0089] Computer assisted Rational Drug Design
[0090] Another way to isolate inhibitors is through rational
design. This is achieved through structural information and
computer modeling. Computer modeling technology allows
visualization of the three-dimensional atomic structure of a
selected molecule and the rational design of new compounds that
will interact with the molecule. The three-dimensional construct
typically depends on data from X-ray crystallographic analyses or
NMR imaging of the selected molecule. The molecular dynamics
require force field data. The computer graphics systems enable
prediction of how a new compound will link to the target molecule
and allow experimental manipulation of the structures of the
compound and target molecule to perfect binding specificity. For
example, using NMR spectroscopy, Inouye and coworkers were able to
obtain the structural information of N-terminal truncated TSHK
(transmembrane sensor histidine kinases) fragments which retain the
structure of the individual sub-domains of the catalytic site of a
TSHK. On the basis of the NMR study, they were able to identify
potential TSHK inhibitors (U.S. Pat. No. 6,077,682 to Inouye, the
teachings of which are incorporated herein in their entirety).
Another good example is based on the three-dimensional structure of
a calcineurin/FKBP12/FK506 complex determined using high resolution
X-ray crystallography to obtain the shape and structure of both the
calcineurin active site binding pocket and the auxiliary
FKBP12/FK506 binding pocket (U.S. Pat. No. 5,978,740 to Armistead,
the teachings of which are incorporated herein in their entirety).
With this information in hand, researchers can have a good
understanding of the association of natural ligands or substrates
with the binding pockets of their corresponding receptors or
enzymes and are thus able to design and make effective
inhibitors.
[0091] Prediction of molecule-compound interaction when small
changes are made in one or both requires molecular mechanics
software and computationally intensive computers, usually coupled
with user-friendly, menu-driven interfaces between the molecular
design program and the user. Examples of molecular modeling systems
are the CHARMm and QUANTA programs, Polygen Corporation, Waltham,
Mass. CHARMm performs the energy minimization and molecular
dynamics functions. QUANTA performs the construction, graphic
modeling and analysis of molecular structure. QUANTA allows
interactive construction, modification, visualization, and analysis
of the behavior of molecules with each other.
[0092] A number of articles review computer modeling of drugs
interactive with specific proteins, such as Rotivinen, et al., 1988
Acta Pharmaceutica Fennica 97, 159-166; Ripka, New Scientist 54-57
(Jun. 16, 1988); McKinaly and Rossmann, 1989 Annu. Rev. Pharmacol.
Toxicol. 29, 111-122; Perry and Davies, QSAR: Quantitative
Structure-Activity Relationships in Drug Design pp. 189-193 (Alan
R. Liss, Inc. 1989; Lewis and Dean, 1989 Proc. R. Soc. Lond. 236,
125-140 and 141-162; and, with respect to a model enzyme for
nucleic acid components, Askew, et al., 1989 J. Am. Chem. Soc. 111,
1082-1090, the teachings of all of which are incorporated herein in
their entirety). Other computer programs that screen and
graphically depict chemicals are available from companies such as
BioDesign, Inc., Pasadena, Calif., Allelix, Inc, Mississauga,
Ontario, Canada, and Hypercube, Inc., Cambridge, Ontario.
[0093] Although described above with reference to design and
generation of compounds which could alter binding, one could also
screen libraries of known compounds, including natural products or
synthetic chemicals, and biologically active materials, including
proteins, for compounds which alter substrate binding or enzymatic
activity.
[0094] Screening of Libraries
[0095] Design of substrate analogs and rational drug design are
based on knowledge of the active site and target, and utilize
computer software programs that create detailed structures of the
enzyme and its substrate, as well as ways they interact, alone or
in the presence of inhibitor. These techniques are significantly
enhanced with X-ray crystallographic data in hand. Inhibitors can
also be obtained by screening libraries of existing compounds for
those which inhibit the catalytically active enzyme. In contrast to
reports in the literature relating to memapsin 2, the enzyme
described herein has activity analogous to the naturally produced
enzyme, providing a means for identifying compounds which inhibit
the endogenous activity. These potential inhibitors are typically
identified using high throughput assays, in which enzyme, substrate
(preferably a chromogenic substrate) and potential inhibitor
(usually screened across a range of concentrations) are mixed and
the extent of cleavage of substrate determined. Potentially useful
inhibitors are those which decrease the amount of cleavage.
[0096] II. Preparation of Catalytically Active Recombinant Memapsin
2
[0097] Cloning and Expression of Memapsin 2
[0098] Memapsin 2 was cloned and the nucleotide (SEQ ID NO: 1) and
predicted amino acid (SEQ ID NO: 2) sequences were determined, as
described in Example 1. The cDNA was assembled from the fragments.
The nucleotide and the deduced protein sequence are shown in SEQ ID
NOS: 1 and 2, respectively. The protein is the same as the aspartic
proteinase 2 (ASP2) described in EP 0 855 444 A by SmithKline
Beecham Pharmaceuticals, (published Jul. 29, 1998), U.S. Pat. No.
6,319,689, Sinha, et al., Nature 402, 537-540 (December 1999) and
Vassar, et al., Science 286, 735-741 (Oct. 22, 1999), the teachings
of all of which are incorporated herein in their entirety).
[0099] Pro-memapsin 2 is homologous to other aspartic proteases,
and shares homology with mouse ASP1 (U.S. Pat. No. 6,291,223, the
teachings of which are incorporated herein in its entirety). Based
on the alignments, Pro-memapsin 2 contains a pro region, an
aspartic protease region, and a trans-membrane region near the
C-terminus. The C-terminal domain is over 80 residues long. The
active enzyme is memapsin 2 and its pro-enzyme is pro-memapsin
2.
[0100] Refolding Catalytically Active Enzyme
[0101] In order to determine the substrate specificity and to
design inhibitors, it is necessary to express catalytically active
recombinant enzyme. Since the active site is not in the
transmembrane region and activity does not require membrane
anchoring, memapsin 2 was expressed in E. coli in two different
lengths, both without the transmembrane region, and purified, as
described in Example 3. The procedures for the culture of
transfected bacteria, induction of synthesis of recombinant
proteins and the recovery and washing of inclusion bodies
containing recombinant proteins are essentially as described by Lin
et al, (1994), the teachings of which are incorporated herein in
its entirety. For refolding, the protein is dissolved in a strong
denaturing/reducing solution such as 8 M urea/100 mM
beta-mercaptoethanol. The rate at which the protein is refolded,
and in what solution, is critical to activity. In one method, the
protein is dissolved into 8 M urea/100 mM beta-mercaptoethanol then
rapidly diluted into 20 volumes of 20 mM-Tris, pH 9.0, which is
then slowly adjusted to pH 8 with 1 M HCl. The refolding solution
is then kept at 4.degree. C. for 24 to 48 hours before proceeding
with purification. In the second method, an equal volume of 20 mM
Tris, 0.5 mM oxidized/1.25 mM reduced glutathione, pH 9.0 is added
to rapidly stirred pro-memapsin 2 in 8 M urea/10 mM
beta-mercaptoethanol. The process is repeated three more times with
1 hour intervals. The resulting solution is then dialyzed against
sufficient volume of 20 mM Tris base so that the final urea
concentration is 0.4 M. The pH of the solution is then slowly
adjusted to 8.0 with 1 M HCl.
[0102] The refolded protein is then further purified by column
chromatography, based on molecular weight exclusion, and/or elution
using a salt gradient, and analyzed by SDS-PAGE analysis under
reduced and non-reduced conditions.
[0103] III. Substrate Specificity and Enzyme Kinetics of Memapsin
2
[0104] The inhibitors can be screened for inhibition of binding and
cleavage by memapsin 2 of its substrate.
[0105] Substrate Specificity
[0106] The presence of memapsin 2 (M2) in the brain indicated that
it might hydrolyze the P-amyloid precursor protein (APP). As
described below, detailed enzymatic and cellular studies
demonstrated that M2 fits all the criteria of the .beta.-secretase.
The M2 three-dimensional structure modeled as a type I integral
membrane protein. The model suggested that its globular protease
unit can hydrolyze a membrane anchored polypeptide at a distance
range of 20-30 residues from the membrane surface. As a
transmembrane protein of the brain, APP is a potential substrate
and its beta-secretase site, located about 28 residues from the
plasma membrane surface, is within in the range for M2
proteolysis.
[0107] A synthetic peptide derived from this site (SEVKM/DAEFR)
(SEQ ID NO: 5) was hydrolyzed by M2.sub.pd (modified M2 containing
amino acids from Ala.sup.-8P to Ala.sup.326) at the beta-secretase
site (marked by /). A second peptide (SEVNL/DAEFR) (SEQ ID NO: 6)
derived from the APP beta-secretase site and containing the
`Swedish mutation` (Mullan, M. et al., Nature Genet. 2:340-342
(1992), the teachings of which are incorporated herein in its
entirety), known to elevate the level of A.beta. production in
cells (Citron, M. et al., Nature 260:672-674 (1992), the teachings
of which are incorporated herein in its entirety), was hydrolyzed
by M2.sub.pd with much higher catalytic efficiency. Both substrates
were optimally cleaved at pH 4.0. A peptide derived from the
processing site of presenilin 1 (SVNM/AEGD) (SEQ ID NO: 7) was also
cleaved by M2.sub.pd with less efficient kinetic parameters. A
peptide derived from the APP gamma-secretase site (KGGVVIATVIVK)
(SEQ ID NO: 8) was not cleaved by M2.sub.pd. Pepstatin A inhibited
M2.sub.pd poorly (IC.sub.50 approximately approximately 0.3 mM).
The kinetic parameters indicate that both presenilin 1 (k.sub.cat,
0.67 s.sup.-1; K.sub.m, 15.2 mM; k.sub.cat/K.sub.m, 43.8
s.sup.-1M.sup.-1) and native APP peptides (k.sub.cat/K.sub.m, 39.9
s.sup.-1M.sup.-1) are not as good substrates as the Swedish APP
peptide (k.sub.cat, 2.45 s.sup.-1,K.sub.m, 1 mM; k.sub.cat/K.sub.m,
2450 s.sup.-1M.sup.-1).
[0108] To determine if M2 possesses an APP beta-secretase function
in mammalian cells, memapsin 2 was transiently expressed in HeLa
cells (Lin, X., et al., FASEB J. 7:1070-1080 (1993), the teachings
of which are incorporated herein in its entirety), metabolically
pulse-labeled with .sup.35S-Met, then immunoprecipitated with
anti-APP antibodies for visualization of APP-generated fragments
after SDS-polyacrylamide electrophoresis and imaging. SDS-PAGE
patterns of immuno-precipitated APP N.beta.-fragment (97 kD band)
from the conditioned media (2 h) of pulse-chase experiments showed
that APP was cleaved by M2. Controls transfected with APP alone and
co-transfected with APP and M2 with Bafilomycin A1 added were
performed. SDS-PAGE patterns of APP .beta.C-fragment (12 kD) were
immunoprecipitated from the conditioned media of the same
experiment as discussed above. Controls transfected with APP alone;
co-transfected with APP and M2; co-transfected with APP and M2 with
Bafilomycin A1; transfections of Swedish APP; and co-transfections
of Swedish APP and M2 were performed. SDS-PAGE gels were also run
of immuno-precipitated M2 (70 kD), M2 transfected cells;
untransfected HeLa cells after long time film exposure; and
endogenous M2 from HEK 293 cells. SDS-PAGE patterns of APP
fragments (100 kD betaN-fragment and 95 kD betaN-fragment)
recovered from conditioned media after immuno-precipitation using
antibodies specific for different APP regions indicated that
memapsin 2 cleaved APP.
[0109] Cells expressing both APP and M2 produced the 97 kD APP beta
N-fragment (from the N-terminus to the beta-secretase site) in the
conditioned media and the 12 kD betaC-fragment (from the
beta-secretase site to the C-terminus) in the cell lystate.
Controls transfected with APP alone produced little detectable
betaN-fragment and no beta C-fragment. Bafilomycin A1, which is
known to raise the intra-vesicle pH of lysosomes/endosomes and has
been shown to inhibit APP cleavage by beta-secretase (Knops, J. et
al., J. Biol. Chem. 270: 2419-2422 (1995), the teachings of which
are incorporated herein in its entirety), abolished the production
of both APP fragments beta N- and beta C- in co-transfected cells.
Cells transfected with Swedish APP alone did not produce the beta
C-fragment band in the cell lysate but the co-transfection of
Swedish APP and M2 did. This Swedish beta C-fragment band is more
intense than that of wild-type APP. A 97-kD beta N-band is also
seen in the conditioned media but is about equal intensity as the
wild-type APP transfection.
[0110] These results indicate that M2 processes the beta-secretase
site of APP in acidic compartments such as the endosomes. To
establish the expression of transfected M2 gene, the pulse-labeled
cells were lysed and immuno-precipitated by anti-M2 antibodies. A
70 kD M2 band was seen in cells transfected with M2 gene, which has
the same mobility as the major band from HEK 293 cells known to
express beta-secretase (Citron, M. et al, Nature 260:672-674
(1992), the teachings of which are incorporated herein in its
entirety). A very faint band of M2 is also seen, after a long film
exposure, in untransfected HeLa cells, indicating a very low level
of endogenous M2, which is insufficient to produce betaN- or
betaC-fragments without M2 transfection. Antibody A.beta..sub.1-17,
which specifically recognizes residues 1-17 in A.beta. peptide, was
used to confirm the correct beta-secretase site cleavage. In cells
transfected with APP and M2, both beta N- and beta N-fragments are
visible using an antibody recognizing the N-terminal region of APP
present in both fragments. Antibody A.beta..sub.1-17 recognize the
beta N-fragment produced by endogenous beta-secretase in the
untransfected cells. This antibody was, however, unable to
recognize the betaN-fragment known to be present in cells
co-transfected with APP and M2. These observations confirmed that
betaN-fragment is the product of beta-secretase site cut by M2,
which abolished the recognition epitope of A.beta..sub.1-17.
[0111] In specificity studies, it was found that M2.sub.pd cleaved
its pro peptide (2 sites) and the protease portion (2 sites) during
a 16 h incubation after activation (Table 1). Besides the three
peptides discussed above, M2.sub.pd also cleaved oxidized bovine
insulin B chain and a synthetic peptide Nch. Native proteins were
not cleaved by M2.sub.pd.
[0112] These same methods can be used in combination with the
disclosed inhibitors to screen for efficacy in inhibiting the
memapsin.
[0113] IV. Methods of diagnosis and treatment
[0114] Inhibitors can be used in the diagnosis and treatment and/or
prevention of Alzheimer's disease and conditions associated
therewith, such as elevated levels of the forty-two amino acid
peptide cleavage product, and the accumulation of the peptide in
amyeloid plaques.
[0115] Diagnostic Uses
[0116] The substrate analogs can be used as reagents for
specifically binding to memapsin 2 or memapsin 2 analogs and for
aiding in memapsin 2 isolation and purification or
characterization, as described in the examples. The inhibitors and
purified recombinant enzyme can be used in screens for those
individuals more genetically prone to develop Alzheimer's
disease.
[0117] Therapeutic Uses
[0118] Recombinant human memapsin 2 cleaves a substrate with the
sequence LVNM/AEGD (SEQ ID NO: 9). This sequence is the in vivo
processing site sequence of human presenilins. Both presenilin 1
and presenilin 2 are integral membrane proteins. They are processed
by protease cleavage, which removes the N terminal sequence from
the unprocessed form. Once processed, presenilin forms a two-chain
heterodimer (Capell et al., J. Biol. Chem. 273, 3205 (1998);
Thinakaran et al., Neurobiol. Dis. 4, 438 (1998); Yu et al,
Neurosci Lett. 2;254(3): 125-8 (1998), the teachings of all of
which are incorporated herein in their entirety), which is stable
relative to the unprocessed presenilins. Unprocessed presenilines
are quickly degraded (Thinakaran et al., J. Biol. Chem. 272, 28415
(1997); Steiner et al., J. Biol. Chem. 273, 32322 (1998), the
teachings of all of which are incorporated herein in their
entirety). It is known that presenilin controls the in vivo
activity of beta-secretase, which in turn cleaves the amyloid
precursor protein (APP) leading to the formation of A.beta.42. The
accumulation of A.beta.42 in the brain cells is known to be a major
cause of Alzheimer's disease (for review, see Selkoe, 1998, the
teachings of which are incorporated herein in its entirety). The
activity of presenilin therefore enhances the progression of
Alzheimer's disease. This is supported by the observation that in
the absence of presenilin gene, the production of A.beta.42 peptide
is lowered (De Strooper et al., Nature 391, 387 (1998), the
teachings of which are incorporated herein in its entirety). Since
unprocessed presenilin is degraded quickly, the processed,
heterodimeric presenilin must be responsible for the accumulation
of A.beta.42 leading to Alzheimer's disease. The processing of
presenilin by memapsin 2 would enhance the production of A.beta.42
and therefore, further the progress of Alzheimer's disease.
Therefore a memapsin 2 inhibitor that crosses the blood brain
barrier can be used to decrease the likelihood of developing or
slow the progression of Alzheimer's disease which is mediated by
deposition of A.beta.42. Since memapsin 2 cleaves APP at the beta
cleavage site, prevention of APP cleavage at the beta cleavage site
will prevent the build up of A.beta.42.
[0119] Pharmaceutically Acceptable Carriers
[0120] The inhibitors will typically be administered orally or by
injection. Oral administration is preferred. Alternatively, other
formulations can be used for delivery by pulmonary, mucosal or
transdermal routes. The inhibitor will usually be administered in
combination with a pharmaceutically acceptable carrier.
Pharmaceutical carriers are known to those skilled in the art. The
appropriate carrier will typically be selected based on the mode of
administration. Pharmaceutical compositions may also include one or
more active ingredients such as antimicrobial agents,
antiinflammatory agents, and analgesics.
[0121] Preparations for parenteral administration or administration
by injection include sterile aqueous or non-aqueous solutions,
suspensions, and emulsions. Examples of non-aqueous solvents are
propylene glycol, polyethylene glycol, vegetable oils such as olive
oil, and injectable organic esters such as ethyl oleate. Aqueous
carriers include water, alcoholic/aqueous solutions, emulsions or
suspensions, including saline and buffered media. Preferred
parenteral vehicles include sodium chloride solution, Ringer's
dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed
oils. Intravenous vehicles include fluid and nutrient replenishers,
and electrolyte replenishers (such as those based on Ringer's
dextrose).
[0122] Formulations for topical (including application to a mucosal
surface, including the mouth, pulmonary, nasal, vaginal or rectal)
administration may include ointments, lotions, creams, gels, drops,
suppositories, sprays, liquids and powders. Formulations for these
applications are known. For example, a number of pulmonary
formulations have been developed, typically using spray drying to
formulate a powder having particles with an aerodynanmic diameter
of between one and three microns, consisting of drug or drug in
combination with polymer and/or surfactant.
[0123] Compositions for oral administration include powders or
granules, suspensions or solutions in water or non-aqueous media,
capsules, sachets, or tablets. Thickeners, flavorings, diluents,
emulsifiers, dispersing aids or binders may be desirable.
[0124] Peptides as described herein can also be administered as a
pharmaceutically acceptable acid- or base- addition salt, formed by
reaction with inorganic acids such as hydrochloric acid,
hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid,
sulfuric acid, and phosphoric acid, and organic acids such as
formic acid, acetic acid, propionic acid, glycolic acid, lactic
acid, pyruvic acid, oxalic acid, malonic acid, succinic acid,
maleic acid, and fumaric acid, or by reaction with an inorganic
base such as sodium hydroxide, ammonium hydroxide, potassium
hydroxide, and organic bases such as mono-, di-, trialkyl and aryl
amines and substituted ethanolamines.
[0125] Dosages
[0126] Dosing is dependent on severity and responsiveness of the
condition to be treated, but will normally be one or more doses per
day, with course of treatment lasting from several days to several
months or until the attending physician determines no further
benefit it if will be obtained. Persons of ordinary skill can
determine optimum dosages, dosing methodologies and repetition
rates.
[0127] The dosage ranges are those large enough to produce the
desired effect in which the symptoms of the memapsin 2 mediated
disorder are alleviated (typically characterized by a decrease in
size and/or number of amyloid plaque, or by a failure to increase
in size or quantity), or in which cleavage of the A.beta.42 peptide
is decreased. The dosage can be adjusted by the individual
physician in the event of any counterindications.
[0128] The present invention will be further illustrated by the
following examples which are not intended to be limiting in any
way.
[0129] Exemplification
EXAMPLE 1
Proteolytic Activity and Cleavage-Site Preferences of Recombinant
Memapsin 2
[0130] The amino acid sequence around the proteolytic cleavage
sites of APP was determined in order to establish the specificity
of memapsin 2. Recombinant pro-memapsin 2-T1 was incubated in 0.1 M
sodium acetate, pH 4.0, for 16 hours at room temperature in order
to create autocatalyzed cleavages. The products were analyzed using
SDS-polyacrylamide gel electrophoresis. Several bands which
corresponded to molecular weights smaller than that of pro-memapsin
2 were observed. The electrophoretic bands were trans-blotted onto
a PVDF membrane. Four bands were chosen and subjected to N-terminal
sequence determination in a Protein Sequencer. The N-terminal
sequence of these bands established the positions of proteolytic
cleavage sites on pro-memapsin 2.
[0131] In addition, the oxidized U-chain of bovine insulin and two
different synthetic peptides were used as substrates for memapsin 2
to determine the extent of other hydrolysis sites. These reactions
were carried out by auto-activated pro-memapsin 2 in 0.1 M sodium
acetate, pH 4.0, which was then incubated with the peptides. The
hydrolytic products were subjected to HPLC on a reversed phase C-18
column and the eluent peaks were subjected to electrospray mass
spectrometry for the determination of the molecular weight of the
fragments. Two hydrolytic sites were identified on oxidized insulin
B-chain (Table1). Three hydrolytic sites were identified from
peptide NCH-gamma. A single cleavage site was observed in synthetic
peptide PS1-gamma, whose sequence (LVNMAEGD) (SEQ ID NO: 10) is
derived from the beta-processing site of human presenilin 1 (Table
1).
2TABLE 1 Substrate Specificity of Memapsin 2 Site # Substrate P4 P3
P2 P1 P1' P2' P3' P4' 1 Pro- R G S M A G V L SEQ ID NO: 11 memapsin
2 (aa 12-18 of SEQ ID NO: 3) 2 G T Q H G I R L SEQ ID NO: 12 (aa
23-30 of SEQ ID NO: 3) 3 S S N F A V G A SEQ ID NO: 13 (aa 98-105
of SEQ ID NO: 3) 4 G L A Y A E I A SEQ ID NO: 14 (aa 183-190 of SEQ
ID NO: 3) 5 Oxidized H L C{circumflex over ( )} G S H L V SEQ ID
NO: 15 6 Insulin B- C{circumflex over ( )} G E R G F F Y
C{circumflex over ( )} is cysteic acid; Chain' SEQ ID NO: 16
C{circumflex over ( )} is cysteic acid 7 Synthetic V G S G V Three
sites cleaved 8 Peptide* V G S G V L L in a peptide: 9 G V L L S R
K VGSGVLLSRK (SEQ ID NO: 30) SEQ ID NO: 17 SEQ ID NO: 18 SEQ ID NO:
19 10 Peptide** L V N M A E G D SEQ ID NO: 10 Positions P.sub.n and
P.sub.n' (wherein n is 1, 2, 3, 4, etc.) refer to the positioning
of the amino acids in a peptide relative to the site of cleavage,
indicated by the double vertical bar. Position numbers increase
distally from the scissile bond. *Synthetic peptide based upon the
amino acid sequence of Notch protein (Accession number AAG33848).
Memapsin 2 cleaves the Notch peptide at three sites. **Synthetic
peptide based upon the processing site of Presenilin-1 (Accession
number P49768, Sherrington et al., Nature 375(6534): 754-760(1995),
the teachings of which are hereby incorporated by reference in its
entirety).
EXAMPLE 2
Activation of Pro-Memapsin 2 and Enzyme Kinetics
[0132] Incubation in 0.1 M sodium acetate, pH 4.0, for 16 h at
22.degree. C. auto-catalytically converted pro-M2.sub.pd to
M2.sub.pd. For initial hydrolysis tests, two synthetic peptides
were separately incubated with pro-M2.sub.pd in 0.1 M Na acetate,
pH 4.0 for different periods ranging from 2 to 18 h. The incubated
samples were subjected to LC/MS for the identification of the
hydrolytic products. For kinetic studies, the identified HPLC
(Beckman System Gold) product peaks were integrated for
quantitation. The K.sub.m and k.sub.cat values for presenilin 1 and
Swedish APP peptides (Table 1) were measured by steady-state
kinetics. The individual K.sub.m and k.sub.cat values for APP
peptide could not be measured accurately by standard methods, so
its k.sub.cat/K.sub.m value was measured by competitive hydrolysis
of mixed substrates against presenilin 1 peptide (Fersht, A.
"Enzyme Structure and Mechanism", 2.sup.nd Ed., W.H. Freeman and
Company, New York. (1985), the teachings of which are incorporated
herein in their entirety).
[0133] The conversion of pro-M2.sub.pd at pH 4.0 to smaller
fragments was shown by SDS-polyacrylamide electrophoresis. The
difference in migration between pro-M2.sub.pd and converted enzyme
is evident in a mixture of the two.
EXAMPLE 3
Design and Synthesis of Memapsin 2 Inhibitors OM99-1 and OM99-2
[0134] Based on the results of specificity studies of memapsin 2,
it was predicted that good residues for positions P1 and P1' would
be Leu and Ala. It was subsequently determined from the specificity
data that P1' preferred small residues, such as Ala and Ser.
However, the crystal structure (determined below) indicates that
this site can accommodate a lot of larger residues. It was
demonstrated that P1' of memapsin 2 is the position with the most
stringent specificity requirement where residues of small side
chains, such as Ala, Ser, and Asp, are preferred. Ala was selected
for P1' mainly because its hydophobicity over Ser and Asp is
favored for the penetration of the blood-brain barrier, a
requirement for the design of a memapsin 2 inhibitor drug for
treating Alzheimer's disease. Therefore, inhibitors were designed
to place a transition-state analogue isostere between Leu and Ala
(shown as Leu*Ala, where * represents the transition-state
isostere, --CH(OH)--CH.sub.2--) and the subsite P4, P3, P2, P2',
P3' and P4' are filled with the beta-secretase site sequence of the
Swedish mutant from the beta-amyloid protein.
[0135] OM99-1: Val-Asn-Leu*Ala-Ala-Glu-Phe (SEQ ID NO: 20)
[0136] OM99-2: Glu-Val-Asn-Leu*Ala-Ala-Glu-Phe (SEQ ID NO: 21)
[0137] The Leu*Ala dipeptide isostere was synthesized as follows:
1
[0138] The Leu-Ala dipeptide isostere for the M.sub.2-inhibitor was
prepared from L-leucine. A bolded number in the following
description, e.g. 2, 3, 4, etc, refers to the respective numbered
compound in synthesis schemes 1, 2, and 3. The bolded number is
also referred to herein as "compound", e.g. compound 2, of the
synthesis scheme.
[0139] As shown in Scheme 1, L-leucine was protected as its
BOC-derivative 2 by treatment with BOC.sub.2O in the presence of
10% NaOH in diethyl ether for 12 h. Boc-leucine 2 was then
converted to Weinreb amide 3 by treatment with isobutyl
chcloroformate and N-methylpiperidine followed by treatment of the
resulting mixed anhydride with N,O-dimethylhydroxylamine (Nahm and
Weinreb, Tetrahedron Letters 1981, 32: 3815, the teachings of which
are incorporated herein in their entirety). Reduction of 3 with
lithium aluminum hydride in diethyl ether provided the aldehyde 4.
Reaction of the aldehyde 4 with lithium propiolate derived from the
treatment of ethyl propiolate and lithium diisopropylamide afforded
the acetylenic alcohol 5 as an inseparable mixture of diastereomers
(5.8: 1) in 42% isolated yield (Fray, Kaye and Kleinman, J. Org.
Chem. 1986, 51: 4828-33, the teachings of which are incorporated
herein in their entirety). Catalytic hydrogenation of 5 over
Pd/BaSO.sub.4 followed by acid-catalyzed lactonization of the
resulting gamma-hydroxy ester with a catalytic amount of acetic
acid in toluene at reflux, furnished the gamma-lactone 6 and 7 in
73% yield.
[0140] The isomers were separated by silica gel chromatography by
using 40% ethyl acetate in hexane as the eluent. Introduction of
the methyl group at C-2 was accomplished by stereoselective
alkylation of 7 with methyl iodide (Scheme 2). Thus, generation of
the dianion of lactone 7 with lithium hexamethyldisilazide (2.2
equivalents) in tetrahydrofuran at -78.degree. C. (30 min) and
alkylation with methyl iodide (1.1 equivalents) for 30 min at
-78.degree. C., followed by quenching with propionic acid (5
equivalents), provided the desired alkylated lactone 8 (76% yield)
along with a small amount (less than 5%) of the corresponding
epimer (Ghosh and Fidanze, 1998 J. Org. Chem. 1998, 63, 6146-54,
the teachings of which are incorporated herein in their entirety).
The epimeric cis-lactone was removed by column chromatography over
silica gel using a mixture (3:1) of ethyl acetate and hexane as the
solvent system. The stereochemical assignment of alkylated lactone
8 was made based on extensive .sup.1H-NMR NOE experiments. Aqueous
lithium hydroxide promoted hydrolysis of the lactone 8 followed by
protection of the gamma-hydroxyl group with tert-butyldimethylsilyl
chloride in the presence of imidazole and dimethylaminopyridine in
2
[0141] dimethylformamide afforded the acid 9 in 90% yield after
standard work-up and chromatography. Selective removal of the
BOC-group was effected by treatment with trifluoroacetic acid in
dichloromethane at 0.degree. C. for 1 h. The resulting amine salt
was then reacted with commercial (Aldrich, Milwaukee)
Fmoc-succinimide derivative in dioxane in the presence of aqueous
NaHCO.sub.3 to provide the Fmoc-protected L*A isostere 10 in 65%
yield after chromatography. Protected isostere 10 was utilized in
the preparation of a random sequence inhibitor library.
[0142] Experimental Procedure
[0143] N-(tert-Butoxycarbonyl)-L-Leucine (Compound 2).
[0144] To the suspension of 10 g (76.2 mmol ) of L-leucine in 140
mL of diethyl ether was added 80 mL of 10% NaOH. After all solid
dissolves, 20 mL (87.1 mmol) of BOC.sub.2O was added to the
reaction mixture. The resulting reaction mixture was stirred at
23.degree. C. for 12 h. After this period, the layers were
separated and the aqueous layer was acidified to pH 1 by careful
addition of 1 N aqueous HCl at 0.degree. C. The resulting mixture
was extracted with ethyl acetate (3.times.100 mL). The organic
layers were combined and washed with brine and dried over anhydrous
Na.sub.2SO.sub.4. The solvent was removed under reduced pressure to
provide title product which was used directly for next reaction
without further purification (yield, 97%). .sup.1H NMR (400 MHz,
CDCl.sub.3) .delta. 4.89 (broad d, 1H, J=8.3 Hz), 4.31 (m, 1H),
1.74-1.49 (m, 3H), 1.44 (s, 9H), 0.95 (d, 6H, J=6.5 Hz).
[0145] N-(tert-Butoxycarbonyl)-L-leucine-N'-methoxy-N'-methyla-mide
(Compound 3).
[0146] To a stirred solution of N,O-dimethylhydroxyamine
hydrochloride (5.52 g, 56.6 mmol) in dry dichloromethane (25 mL)
under N.sub.2 atmosphere at 0.degree. C., -methylpiperidine (6.9
mL, 56.6 mmol) was added dropwise. The resulting mixture was
stirred at 0.degree. C. for 30 min. In a separate flask,
N-(tert-butyloxycarbonyl)-L-leucine (2) (11.9 g, 51.4 mmol) was
dissolved in a mixture of THF (45 mL) and dichloromethane (180 mL)
under N.sub.2 atmosphere. The resulting solution was cooled to
-20.degree. C. To this solution was added 1-methylpiperidine (6.9
mL, 56.6 mmol) followed by isobutyl chloroformate (7.3 mL, 56.6
mmol). The resulting mixture was stirred for 5 minutes at
-20.degree. C. and the above solution of N,O-dimethylhydroxyamine
was added to it. The reaction mixure was kept -20.degree. C. for 30
minutes and then warmed to 23.degree. C. The reaction was quenched
with water and the layers were separated. The aqueous layer was
extracted with dichloromethane (3.times.100 mL). The combined
organic layers were washed with 10% citric acid, saturated sodium
bicarbonate, and brine. The organic layer was dried over anhydrous
Na.sub.2SO.sub.4 and concentrated under the reduced pressure. The
residue was purified by flash silica gel chromatography (25% ethyl
acetate/hexane) to yield the title compound 3 (13.8 g, 97%) as a
pale yellow oil. .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 5.06
(broad d, 1H, J=9.1 Hz), 4.70 (m, 1H), 3.82 (s, 3H), 3.13 (s, 3H),
1.70 (m, 1H), 1.46-1.36 (m, 2H) 1.41 (s, 9H), 0.93 (dd, 6H, J=6.5,
14.2 Hz).
[0147] N-(tert-Butoxycarbonyl)-L-leucinal (Compound 4).
[0148] To a stirred suspension of lithium aluminum hydride (770 mg,
20.3 mmol) in dry diethyl ether (60 mL) at -40.degree. C. under
N.sub.2 atmosphere, was added
N-tert-butyloxycarbonyl-L-leucine-N'-methoxy-N'-met- hylamide (5.05
g, 18.4 mmol) in diethyl ether (20 mL). The resulting reaction
mixture was stirred for 30 min. After this period, the reaction was
quenched with 10% NaHSO.sub.4 solution (30 mL). The resulting
reaction mixture was then warmed to 23.degree. C. and stirred at
that temperature for 30 min. The resulting solution was filtered
and the filter cake was washed by two portions of diethyl ether.
The combined organic layers were washed with saturated sodium
bicarbonate, brine and dried over anhydrous MgSO.sub.4. Evaporation
of the solvent under reduced pressure afforded the title aldehyde 4
(3.41 g) as a pale yellow oil. The resulting aldehyde was used
immediately without further purification. .sup.1H NMR (400 MHz,
CDCl.sub.3) .delta. 9.5 (s, 1H), 4.9 (s, 1H), 4.2 (broad m, 1H),
1.8-1.6 (m, 2H), 1.44 (s, 9H), 1.49-1.39 (m, 1H), 0.96 (dd, 6H,
J=2.7, 6.5 Hz).
[0149] Ethyl (4S,5S)- and
(4R,5S)-5-[(tert-Butoxycarbonyl)amino]-4-hydroxy-
-7-methyloct-2-ynoate (Compound 5).
[0150] To a stirred solution of diisopropylamine (1.1 mL, 7.9 mmol)
in dry THF (60 mL) at 0.degree. C. under N.sub.2 atmosphere, was
added n-BuLi (1.6 M in hexane, 4.95 mL, 7.9 mmol) dropwise. The
resulting solution was stirred at 0.degree. C. for 5 min and then
warmed to 23.degree. C. and stirred for 15 min. The mixture was
cooled to -78.degree. C. and ethyl propiolate (801 mL) in THF (2
mL) was added dropwise over a period of 5 min. The mixture was
stirred for 30 min, after which N-Boc-L-leucinal 4 (1.55 g, 7.2
mmol) in 8 mL of dry THF was added. The resulting mixture was
stirred at -78.degree. C. for 1 h. After this period, the reaction
was quenched with acetic acid (5 mL) in THF (20 mL). The reaction
mixure was warmed up to 23.degree. C. and brine solution was added.
The layers were separated and the organic layer was washed with
saturated sodium bicarbonate and dried over Na.sub.2SO.sub.4.
Evaporation of the solvent under reduced pressure provided a
residue which was purified by flash silica gel chromatography (15%
ethyl acetate/hexane) to afford a mixture (3:1) of acetylenic
alcohols 5 (0.96 g, 42%). .sup.1H NMR (300 MHz, CDCl.sub.3) .delta.
4.64 (d, 1H, J=9.0 Hz), 4.44 (broad s, 1H), 4.18 (m, 2H ), 3.76 (m,
1H), 1.63 (m, 1H), 1.43-1.31 (m, 2H), 1.39 (s, 9H), 1.29-1.18 (m,
3H), 0.89 (m, 6H).
[0151]
(5S,1'S)-5-[1'-[(tert-Butoxycarbonyl)amino]-3'-methylbutyl]-dihydro-
furan-2(3H)-one (Compound 7).
[0152] To a stirred solution of the above mixture of acetylenic
alcohols (1.73 g, 5.5 mmol ) in ethyl acetate (20 mL) was added 5%
Pd/BaSO.sub.4 (1 g). The resulting mixture was hydrogenated at 50
psi for 1.5 h. After this period, the catalyst was filtered off
through a plug of Celite and the filtrate was concentrated under
reduced pressure. The residue was dissolved in toluene (20 mL) and
acetic acid (100 .mu.L). The reaction mixure was refluxed for 6 h.
After this period, the reaction was cooled to 23.degree. C. and the
solvent was evaporated to give a residue which was purified by
flash silica gel chromatography (40% diethyl ether/hexane) to yield
the (5S,1S')-gamma-lactone 7 (0.94 g, 62.8 and the
(5R,1S')-gamma-lactone 6 (0.16 g, 10.7%). Lactone 7: .sup.1H NMR
(400 MHz, CDCl.sub.3) .delta. 4.50-4.44 (m, 2H), 3.84-3.82 (m, 1H),
2.50 (t, 2H, J=7.8 Hz), 2.22-2.10 (m, 2H), 1.64-1.31 (m, 3H), 1.41
(s, 9H), 0.91 (dd, 6H, J=2.2, 6.7 Hz); .sup.13C NMR (75 MHz,
CDCl.sub.3) 6177.2, 156.0, 82.5, 79.8, 51.0, 42.2, 28,6, 28.2,
24.7, 24.2, 23.0, 21.9.
[0153]
(3R,5S,1'S)-5-[1'-[(tert-Butoxycarbonyl)amino)]-3'-methylbutyl]-3-m-
ethyl Dihydrofuran-2(3H)-one (Compound 8).
[0154] To a stirred solution of the lactone 7 (451.8 mg, 1.67 mmol)
in dry THF (8 mL) at -78.degree. C. under N.sub.2 atmosphere, was
added lithium hexamethyldisilazane (3.67 mL, 1.0 M in THF) over a
period of 3 min. The resulting mixture was stirred at -78.degree.
C. for 30 min to generate the lithium enolate. After this period,
MeI (228 .mu.L) was added dropwise and the resulting mixture was
stirred at -78.degree. C. for 20 min. The reaction was quenched
with saturated aqueous NH.sub.4Cl solution and was allowed to warm
to 23.degree. C. The reaction mixture was concentrated under
reduced pressure and the residue was extracted with ethyl acetate
(3.times.100 mL). The combined organic layers were washed with
brine and dried over anhydrous Na.sub.2SO.sub.4. Evaporation of the
solvent afforded a residue which was purified by silica gel
chromatography (15% ethyl acetate/hexane) to furnish the alkylated
lactone 8 (0.36 g, 76%) as an amorphous solid. .sup.1H NMR (300
MHz, CDCl.sub.3) .delta. 4.43 (broad t, 1H, J=6.3 Hz), 4.33 (d, 1H,
J=9.6 Hz), 3.78 (m, 1H), 2.62 (m, 1H), 2.35 (m, 1H), 1.86 (m, 1H),
1.63-1.24 (m, 3H), 1.37 (s, 9H), 1.21 (d, 3H, J=7.5 Hz), 0.87 (dd,
6H, J=2.6, 6.7 Hz); .sup.13C NMR (75 MHz, CDCl.sub.3) .delta.
180.4, 156.0, 80.3, 79.8, 51.6, 41.9, 34.3, 32.5, 28.3, 24.7, 23.0,
21.8, 16.6.
[0155]
(2R,4S,5S)-5-[(tert-Butoxycarbonyl)amino]-4-[(tert-butyldimethylsil-
yl)oxy]-2,7-dimethyloctanoic Acid (Compound 9)
[0156] To a stirred solution of lactone 8 (0.33 g, 1.17 mmol) in
THF (2 mL) was added 1 N aqueous LiOH solution (5.8 mL). The
resulting mixture was stirred at 23.degree. C. for 10 h. After this
period, the reaction mixture was concentrated under reduced
pressure and the remaining aqueous residue was cooled to 0.degree.
C. and acidified with 25% citric acid solution to pH 4. The
resulting acidic solution was extracted with ethyl acetate
(3.times.50 mL). The combined organic layers were washed with
brine, dried over Na.sub.2SO.sub.4 and concentrated to yield the
corresponding hydroxy acid (330 mg) as a white foam. This hydroxy
acid was used directly for the next reaction without further
purification.
[0157] To the above hydroxy acid (330 mg, 1.1 mmol) in anhydrous
DMF was added imidazole (1.59 g, 23.34 mmol) and
tert-butyldimethylchlorosilane (1.76 g, 11.67 mmol). The resulting
mixture was stirred at 23.degree. C. for 24 h. After this period,
MeOH (4 mL) was added and the mixture was stirred for 1 h. The
mixure was diluted with 25% citric acid (20 mL) and was extracted
with ethyl acetate (3.times.20 mL). The combined extracts were
washed with water, brine and dried over anhydrous Na.sub.2SO.sub.4.
Evaporation of the solvent gave a viscous oil which was purified by
flash chromatography over silica gel (35% ethyl acetate/hexane) to
afford the silyl protected acid 9 (0.44 g, 90%). IR (neat)
3300-3000 (broad), 2955, 2932, 2859, 1711 cm.sup.-1; .sup.1H NMR
(400 MHz, DMSO-d.sup.6, 343 K) delta 6.20 (broad s, 1H), 3.68 (m,
1H), 3.51 (broad s, 1H), 2.49-2.42 (m, 1H), 1.83 (t, 1H, J=10.1
Hz), 1.56 (m, 1H), 1.37 (s, 9H), 1.28-1.12 (m, 3H), 1.08 (d, 3H,
J=7.1 Hz), 0.87 (d, 3H, J=6.1 Hz) 0.86 (s, 9H), 0.82 (d, 3H, J=6.5
Hz), 0.084 (s, 3H), 0.052 (s, 3H).
[0158]
(2R,4S,5S)-5-[fluorenylmethyloxycarbonyl)amino]-4-[(tert-butyldi-me-
thyl silyl)oxy]-2,7-dimethyloctanoic Acid (Compound 10).
[0159] To a stirred solution of the acid 9 (0.17 g, 0.41 mmol) in
dichloromethane (2 mL) at 0.degree. C. was added trifluoroacetic
acid (500 .mu.L). The resulting mixture was stirred at 0.degree. C.
for 1 h and an additional portion (500 .mu.L) of trifluoroacetic
acid was added to the reaction mixture. The mixture was stirred for
an additional 30 min and the progress of the reaction was monitored
by TLC. After this period, the solvents were carefully removed
under reduced pressure at a bath temperature not exceeding
5.degree. C. The residue was dissolved in dioxane (3 mL) and
NaHCO.sub.3 (300 mg) in 5 mL of H.sub.2O. To this solution was
added Fmoc-succinimide (166.5 mg, 0.49 mmol) in 5 mL of dioxane.
The resulting mixture was stirred at 23.degree. C. for 8 h. The
mixure was then diluted with H.sub.2O (5 mL) and acidified with 25%
aqueous citric acid to pH 4. The acidic solution was extracted with
ethyl acetate (3.times.50 mL). The combined extracts were washed
with brine, dried over Na.sub.2SO.sub.4 and concentrated under
reduced pressure to give a viscous oil residue. Purification of the
residue by flash chromatography over silica gel afforded the
Fmoc-protected acid 10 (137 mg, 61%) as a white foam. .sup.1H NMR
(400 MHz, DMSO-d.sup.6, 343 K) .delta. 7.84 (d, 2H, J=7.4 Hz), 7.66
(d, 2H, J=8 Hz), 7.39 (t, 2H, J=7.4 Hz), 7.29 (m, 2H), 6.8 (s, 1H),
4.29-4.19 (m, 3H), 3.74-3.59 (m, 2H), 2.49 (m, 1H), 1.88 (m, 1H),
1.58 (m, 1H), 1.31-1.17 (m, 3H), 1.10 (d, 3H, J=7.1 Hz), 0.88 (s,
9H), 0.82 (d, 6H, J=6.2 Hz), 0.089 (s, 3H), 0.057 (s, 3H).
[0160] The synthesis of OM99-1 and OM99-2 were accomplished using
solid state peptide synthesis procedure in which Leu*Ala was
incorporated in the fourth step. The synthesized inhibitors were
purified by reverse phase HPLC and their structure confirmed by
mass spectrometry.
EXAMPLE 4
Design, Synthesis and Analysis of Additional Inhibitors.
[0161] Various substrate analogues other than OM99-1 and OM99-2 can
be similarly designed and synthesized. For example, sixty six
additional inhibitor analogues, MMI-001 to MMI-062, MMI-065,
MMI-066, MMI-070 and MMI-071, all of which resemble an isostere of
the active site of memapsin 2, were designed. The synthesis of the
additional substrate analogues follows that for OM99-1 and OM99-2.
The chemical structures of the additional substrate analogues are
listed in Table 3. The general synthesis of various inhibitors are
outlined in Scheme 3. 3
[0162] As scheme 3 demonstrates, using a standard peptide coupling
procedure, valine derivative 11 was reacted with the dipeptide
isostere 9 in the presence of
N-ethyl-N'-(dimethylaminopropyl)carbodiimide hydrochloride,
triethylamine and 1-hydroxybenzotriazole hydrate in a mixture of
DMF and CH.sub.2Cl.sub.2 to afford the amide derivative 12. Removal
of the silyl protecting group by treatment with tetrabutylammonium
fluoride in THF afforded inhibitor 13 (MMI-001). Exposure of 13 to
trifluoroacetic acid in CH.sub.2Cl.sub.2 resulted in the removal of
BOC group. Coupling of the resulting amine with BOC-asparagine
provided inhibitor 14 (MMI-01 1). Treatment of 14 with
trifluoroacetic acid and coupling of the resulting amine with
BOC-valine under standard conditions afforded inhibitor 16
(MMI-012). For the synthesis of inhibitor MMI-15, compound 13 was
reacted with trifluoroacetic acid and the resulting amine was
coupled with BOC-methionine to afford the inhibitor 15 (MMI-015).
Removal of the BOC group of 16 and coupling of the resulting amine
with BOC-valine provided the inhibitor 17 (MMI-017).
[0163] Preparation of inhibitors MMI-001, MMI-011, MMI-012, MMI-015
and MMI-17:
[0164] Preparation of Valine Derivative (Compound 11):
[0165] N-Boc Valine (500 mg. 2.30 mmol) and benzylamine (0.50 mL,
4.60 mmol) were dissolved in CH.sub.2Cl.sub.2 (20 mL) and DMF (2
mL). To this solution, HOBt (373 mg, 2.76 mmol) and EDC (529 mg,
2.76 mmol), and diisopropylethylamine (2.4 mL, 13.80 mmol) were
added successively at 0.degree. C. After the addition, the reaction
mixture was allowed to warm up to 23.degree. C. and stirred
overnight. The mixture was poured into sat. NaHCO.sub.3 (aq). The
resultant mixture was extracted with 30% EtOAc/hexane. The organic
layer was washed with brine and dried over Na.sub.2SO.sub.4.
Evaporation of the solvent under the reduced pressure gave a
residue which was purified by flash column chromatography (30%
EtOAc/hexane) to give 442 mg (63%) of coupling product. The
resulting amine was dissolved in CH.sub.2Cl.sub.2 (20 mL). TFA (4
mL) was then added at room temperature. The reaction mixture was
stirred for 0.5 hr. and it was concentrated under the reduced
pressure. The amine 11 was obtained quantitative yield. .sup.1H NMR
(500 MHz, CDCl.sub.3) 0.87 (3H, d, J=6.9 Hz), 1.02 (3H, d, J=6.9
Hz), 2.00 (2H, br s), 2.37 (1H, m), 3.36 (1H, br s), 4.43-4.52 (2H,
m), 7.27-7.37 (5H, m), 7.70 (1H, br s).
[0166] Preparation of Amide Derivative (Compound 12):
[0167] Dipeptide isostere 9 (41 mg. 0.10 mmol) and amine 11 (41 mg.
0.20 mmol) were dissolved in DMF (2.0 mL). To this solution, HOBt
(20 mg, 0.15 mmol) and EDC (29 mg, 0.15 mmol), and
diisopropylethylamine (0.2 mL) were added successively at 0.degree.
C. After the addition, the reaction mixture was allowed to warm up
to 23.degree. C. and was stirred overnight. The mixture was poured
into sat. NaHCO.sub.3 (aq). The mixture was extracted with 30%
EtOAc/hexane. The organic layer was washed with brine and dried
over Na.sub.2SO.sub.4. Evaporation of the solvent under the reduced
pressure gave a residue which was purified by column chromatography
(20% EtOAc/hexane) to give 55 md (95%) of amide 12. .sup.1H NMR
(500 MHz, CDCl.sub.3) 0.09 (3H, s), 0.10 (3H, s), 0.91 (9H, s),
0.92-0.98 (12H, m), 1.10 (3H, d, J=6.7 Hz), 1.25 (1H, m), 1.44 (1H,
m), 1.46 (9H, s), 1.63 (1H, m), 1.74 (1H, br s), 1.80 (1H, m), 2.18
(1H, m), 2.56 (1H, m), 3.62-3.78 (2H, m), 4.13 (1H, m), 4.48-4.56
(3H, m), 6.35 (1H, br d, J=8.5 Hz), 6.41 (1H, br s), 7.26-7.40 (5H,
m).
[0168] Preparation of Inhibitor MMI-001 (Compound 13):
[0169] To a solution of amide 12 (61 mg. 0.10 mmol) in THF (1.0 mL0
was added TBAF (1.0 M in THF:0.3 mL, 0.30 mmol) at 23.degree. C.
and it was stirred overnight. The reaction mixture was concentrated
under reduced pressure. The residue was purified by column
chromatography (40% EtOAc/hexane) to give MMI-001 (13, 41 mg, 83%).
.sup.1H NMR (300 MHz, CDCl.sub.3) 0.88-0.98 (12H, m), 1.15 (3H, d,
J=6.9 Hz), 1.40-1.80 (5H, m), 1.43 (9H, s), 2.10 (1H, m), 2.60 (1H,
m), 3.40-3.60 (2H, m), 4.00 (1H, m), 4.20-4.45 (3H, m), 4.70 (1H,
m), 708-718 (5H, m).
[0170] Preparation of Inhibitor MMI-011 (Compound 14):
[0171] To a solution of 13 (44 mg, 0.089 mmol) in CH.sub.2Cl.sub.2
(1 mL) was added TFA (0.2 ml) at 23.degree. C. After 0.5 hr, the
reaction mixture was concentrated under reduced pressure. The
residue was dissolved in DMF (4 mL). To this solution,
N-Boc-asparagine (41 mg, 0.18 mmol), HOBt (24 mg, 0.18 mmol) and
EDC (34 mg 0.18 mmol), and diisopropylethylamine (0.2 mL) were
added successively at 0.degree. C. After the addition, the raction
mixture was allowed to warn up to 23.degree. C. and was stirred
overnight. The mixture was poured into sat. NaHCO.sub.3 (aq). The
mixture was extracted with EtOAc. The organic layer was washed with
brine and dried over Na.sub.2SO.sub.4. Evaporation of the solvent
under the reduced pressure gave a residue which was purified by
column chromatography (4% MeOH/EtOAc) to give 12.5 mg (23%) of
MMI-011 (14). .sup.1H NMR (500 MHz, CD.sub.3OD) 0.85-1.00 (12H, m),
1.10 (3H, d, J=6.7 Hz), 1.20-1.50 (5H, m), 1.45 (9H, s), 1.60 (1H,
m), 1.75 (1H, m), 2.05 (1H, m), 2.50-2.70 (3H, m), 3.45 (1H, m),
3.75 (1H, m), 4.10 (1H, m), 4.30-4.40 (3H, m), 7.20-7.30 (5H,
m).
[0172] Preparation of Inhibitor MMI-015 (Compound 15):
[0173] To a solution of compound 13 (64 mg. 0.13 mmol) in
CH.sub.2Cl.sub.2 (4 mL) was added TFA (1 ml) at 23.degree. C. After
0.5 hr, the reaction mixture was concentrated under reduced
pressure. The residue was dissolved in DMF (4 mL). To this
solution, N-Boc-methionine (65 mg, 0.26 mmol), HOBt (35 mg. 0.26
mmol) and EDC (50 mg, 0.26 mmol), and diisopropylethylamine (0.4
mL) were added successively at 0.degree. C. After the addition, the
reaction mixture was allowed to warm up to 23.degree. C. and was
stirred overnight. The mixture was poured into sat. NaHCO3 (aq).
The mixture was extracted with EtOAc. The organic layer was washed
with brine and dried over Na.sub.2SO.sub.4. Evaporation of the
solvent under the reduced pressure gave a residue which was
purified by column chromatography (80% EtOAc/liexane) to give 37.2
mg (46%) of MMI-015 (15). .sup.1H NMR (300 MHz, CD3OD) 0.80-1.00
(12H, m), 1.50 (3H, d, J=6.7 Hz), 1.20-2.10 (10H, m), 1.45 (9H, s),
2.10 (3H, s), 2.45-2.60 (2H, m), 2.70 (1H, m), 3.50 (1H, m), 3.90
(1H, m), 4.10-4.15 (2H, m), 4.30-4.42 (2H, m), 7.20-7.35 (5H,
m).
[0174] Preparation of Inhibitor MMI-012 (Compound 16):
[0175] To a solution of 13 (8.4 mg, 0.014 mmol) in CH.sub.2Cl.sub.2
(1 mL) was added TFA (0.2 ml) at 23.degree. C. After 0.5 hr, the
reaction mixture was concentrated under reduced pressure. The
residue was dissolved in DMF (1 mL). To this solution, N-Boc-valine
(6 mg, 0.028 mmol), HOBt (3.8 mg, 0.028 mmol) and EDC (5.3 mg.
0.028 mmol), and diisopropylethylamine (0.2 mL) were added
successively at 0.degree. C. After the addition, the reaction
mixture was allowed to warm up to 23.degree. C. and was stirred
overnight. The mixture was poured into sat. NaHCO.sub.3 (aq). The
mixture was extracted with EtOAc. The organic layer was washed with
brine and dried over Na.sub.2SO.sub.4. Evaporation of the solvent
under reduced pressure gave a residue which was purified by column
chromatography (4% MeOH/EtOAc) to give 2.1 mg (21%) of inhibitor
MMI-012 (16). .sup.1H NMR (400 MHz, CD.sub.3OD) 0.80-1.00 (18H, m),
1.05 (3H, d, J=6.7 Hz), 1.10-1.50 (6H, m), 1.40 (9H, s), 1.55 (1H,
m), 1.70 (1H, m), 2.00 (1H, m), 2.45-2.70 (3H, m), 3.45 (1H, m),
3.80-4.45 (6H, m), 7.15-7.30 (5H, m).
[0176] Preparation of Inhibitor MMI-107 (Compound 17):
[0177] To a solution of 15 (14.5 mg, 0.023 mmol) in
CH.sub.2Cl.sub.2 (2 mL) was added TFA (0.5 ml) at 23.degree. C.
After 0.5 hr. the reaction mixture was concentrated under reduced
pressure. The residue was dissolved in DMF (2 mL). To this
solution, N-Boc-valine (10 mg., 0.046 mmol), HOBt (6.2 mg, 0.046
mmol) and EDC (8.8 mg, 0.046 mmol), and diisopropylethylamine (0.4
mL) were added successively at 0.degree. C. After the addition, the
reaction mixture was allowed to warm up to 23.degree. C. and was
stirred overnight. The mixture was poured into sat. NaHCO.sub.3
(aq). The mixture was extracted with EtOAc. The organic layer was
washed with brine and dried over Na.sub.2SO.sub.4. Evaporation of
the solvent under the reduced pressure gave a residue which was
purified by column chromatography (EtOAc) to give 5.5 mg (33%) of
MMI-017 (17). Rf=(10% EtOAc/hexane); .sup.1H NMR (300 MHz,
CD.sub.3OD) 0.80-1.00 (18H, m), 1.10 (3H, d, J=6.7 Hz), 1.20-2.10
(11H, m), 1.43 (9H, s), 2.10 (3H, s), 2.40-2.60 (2H, m), 2.70 (1H,
m), 3.50 (1H, m), 3.80-3.95 (2H, m), 4.18 (1H, m), 4.30-4.50 (3H,
m), 7.20-7.30 (5H, m).
[0178] Enzyme activity of the isosteres was measured as described
above, but with the addition of either OM99-1, OM99-2 or one of
MMI-001-MMI-062, MMI-065, MMI-066, MMI-070 and MMI-071. OM99-1
inhibited recombinant memapsin with a K.sub.i calculated as
3.times.10.sup.-8 M. The substrate used was a synthetic fluorogenic
peptide substrate. The inhibition of OM99-2 on recombinant memapsin
2 was measured using the same fluorogenic substrate. The K.sub.i
value was determined to be 9.58.times.10.sup.-9 M.
[0179] The residues in P1 and P1' are very important since the M2
inhibitor must penetrate the blood-brain barrier (BBB). The choice
of Ala in P1' facilitates the penetration of BBB. Analogues of Ala
side chains will also work. For example, in addition to the methyl
side chain of Ala, substituted methyl groups and groups about the
same size like methyl or ethyl groups can be substituted for the
Ala side chain. Leu at P1 can also be substituted by groups of
similar sizes or with substitutions on Leu side chain. For
penetrating the BBB, it is desirable to make the inhibitors
smaller. One can therefore use OM99-1 as a starting point and
discard the outside subsites P4, P3, P3' and P4'. The retained
structure Asn-Leu*Ala-Ala is then further evolved with
substitutions for a tight-binding M2 inhibitor which can also
penetrate the BBB.
[0180] The other substrate analogues, MMI-001 to MMI-071, were also
tested for enzyme inhibition. For example, the K.sub.i values for
MMI-017, MMI-070 and MMI-071 are comparable to that for OM99-2,
indicating that they are also excellent memapsin inhibitors. The
K.sub.i value for MMI-012, MMI-018, MMI-026, or MMI-066 is
approximately one magnitude higher than that for OM99-2, indicating
that they are competent candidates for memapsin inhibition. The
K.sub.i value for each of the additional substrate analogues and
its corresponding chemical structure are listed in Table 3.
3TABLE 3 Chemical Structures and K.sub.i Values of Additional
Substrate Analogues Comp No./ Structure K.sub.i (nM) 4 MMI-001/
3,738,100.0 5 MMI-002/ 430,940.0 6 MMI-003/ 2,617,000.0 7 MMI-004/
22,423.0 8 MMI-005/ 61.4 9 MMI-006/ 63,288.0 10 MMI-007/ 49,877.0
11 MMI-008/ 73.6 12 MMI-009/ 89.9 13 MMI-010/ 29,403.0 14 MMI-011/
3,134.0 15 MMI-012/ 5.9 16 MMI-013/ 184,160.0 17 MMI-014/ 2,777.8
18 MMI-015/ 5,808.0 19 MMI-016/ 5,640.0 20 MMI-017/ 2.5 21 MMI-018/
8.0 22 MMI-019/ 14,500.0 23 MMI-020/ 1,605.2 24 MMI-021/ 112.2 25
MMI-022/ 11,766.0 26 MMI-023/ 15,273.0 27 MMI-024/ 1129.0 28
MMI-025/ 50.1 29 MMI-026/ 9.4 30 MMI-027/ 11,547.3 31 MMI-028/
9,867.0 32 MMI-029/ 1,696.6 33 MMI-030/ 8,517.0 34 MMI-031/ 863.7
35 MMI-032/ 1,341.0 36 MMI-033/ 16,300.0 37 MMI-034/ 228.8 38
MMI-035/ 426.3 39 MMI-036/ no inhibition 40 MMI-037/ 3.9 41
MMI-038/ 106.0 42 MMI-039/ 1.4 43 MMI-040/ 2.1 44 MMI-041/ no
inhibition 45 MMI-042/ 36,484.1 46 MMI-043/ 49,188.9 47 MMI-044/ no
inhibition 48 MMI-045/ no inhibition 49 MMI-046/ 149,900,000.0 50
MMI-047/ 110,600,000.0 51 MMI-048/ 54,8000,000.0 52 MMI-049/
198,000.0 53 MMI-050/ 146,000.0 54 MMI-051/ no inhibition 55
MMI-052/ 5,790.0 56 MMI-053/ no inhibition 57 MMI-054/ 469,000.0 58
MMI-055/ 631,000.0 59 MMI-056/ 205,000.0 60 MMI-057/ 73,300.0 61
MMI-058/ 408,000.0 62 MMI-059/ 66,300.0 63 MMI-060/ 100,000.0 64
MMI-061/ 15,460,000.0 65 MMI-062/ no inhibition 66 MMI-065/
40.0(97.4) 67 MMI-066/ 21.2 68 MMI-070/ 2.0(4.9) 69 MMI-071/
4.0(1.9)
EXAMPLE 5
Sub-site Specificity of Memapsin 2 (.beta.-secretase)
[0181] The results obtained from OM99-2 demonstrate that by evoking
all eight sub-sites with OM99-2, a high inhibition potency
(K.sub.i=1.6 nM) can be achieved. However, clinically useful
memapsin 2 inhibitors must be small (i.e., typically less than
500-700 daltons) in order to penetrate the blood-brain barrier to
reach the brain. Detailed information on sub-site specificity of
memapsin 2 would provide a better understanding for the design of
small yet tight-binding inhibitors. The complete sub-site
preference of memapsin 2 has been determined based on results from
both substrate kinetics and the probing of random sequence
inhibitor library.
[0182] Experimental Procedures
[0183] The Design of the Defined Substrate Mixtures
[0184] Peptide mixtures were synthesized to probe each of 8
positions in the template sequence EVNLAAEF (SEQ ID NO: 22),
derived from the .beta.-secretase cleavage site in APP and memapsin
2 inhibitor OM99-2 described above. For characterization of each
position, an equimolar mixture of 7 amino acid derivatives were
added in the appropriate cycle of solid phase synthesis (Research
Genetics, Invitrogen, Huntsville, Ala.) resulting in a mixture of 7
peptides, differing by 1 of 7 amino acids at a single position. To
facilitate the rapid quantitation of substrates and hydrolytic
products, high throughput MALDI-TOF MS was used. Therefore, it was
not possible to subsitute allcommon amino acid side chains (19
total, excluding cysteine) in a single mixture due to the identical
masses of some amino acids which would prevent their
identification. Therefore, the different amino acid substitutions
were divided into three groups (Tables 4 and 5) to maximize their
differences in mass and the full set of 19 varied amino acids (less
cysteine) at each subsite were contained in 3 substrate mixtures in
which the peptides were synthesized to incorporate each amino acid
in a single position. A standard peptide was included in each group
for the purpose of relating the quantitative information between
mixtures, for calculation of relative k.sub.cat/K.sub.m. The
substrate of known k.sub.cat/K.sub.m value serves as an internal
standard for normalization of relative initial rates and the
calculation of k.sub.cat/K.sub.m value of other substrates. For
positions P.sub.1', P.sub.2', P.sub.3', and P.sub.4', the template
sequence was extended by 4 residues at the C-terminus (template
EVNLAAEFWHDR, SEQ ID NO: 23, Table 4) to facilitate their detection
in MALDI-TOF MS. Four additional residues were likewise added on
the N-terminus in the template to characterize specificity for
positions P.sub.1, P.sub.2, P.sub.3 and P.sub.4 (template
RWHHEVNLAAEF, SEQ ID NO: 24, Table 5).
4TABLE 4 Peptide Substrate Mixture for Specificity Determination of
Memapsin 2 to the P.sub.1' Position SEQ ID Amino Acid Peptide
Sequence NO. Substrate A (alanine) EVNLAAEFWHDR 25 mixture 1 D
(aspartic acid) EVNLDAEFWHDR 26 S (serine) EVNLSAEFWHDR 27 T
(threonine) EVNLTAEFWHDR 28 I (isoleucine) EVNLIAEFWHDR 29 E
(glutamic acid) EVNLEAEFWHDR 30 F (phenylalanine) EVNLFAEFWHDR 31
Substrate A (alanine) EVNLAAEEWHDR 32 mixture 2 G (glycine)
EVNLGAEFWHDR 33 V (valine) EVNLVAEFWHDR 34 L (leucine) EVNLLAEFWHDR
35 K (lysine) EVNLKAEFWHDR 36 R (arginine) EVNLRAEFWHDR 37 W
(tryptophan) EVNLWAEFWHDR 38 Substrate A (alanine) EVNLAAEFWHDR 39
mixture 3 P (proline) EVNLPAEFWHDR 40 N (asparagine) EVNLNAEFWHDR
41 Q (glutamine) EVNLQAEFWHDR 42 M (methionine) EVNLMAEFWHDR 43 H
(histidine) EVNLHAEFWHDR 44 Y (tyrosine) EVNLYAEFWHDR 45
[0185]
5TABLE 5 Peptide Substrate Mixture for Specificity Determination of
Memapsin 2 to the P.sub.1 Position SEQ ID Amino Acid Peptide
Sequence NO. Substrate F (phenylalanine) RWHHEVNFAAEF 46 mixture 1
D (aspartic acid) RWHHEVNDAAEF 47 S (serine) RWHHEVNSAAEF 48 T
(threonine) RWHHEVNTAAEF 49 I (isoleucine) RWHHEVNIAAEF 50 E
(glutamic acid) RWHHEVNEAAEF 51 G (glycine) RWHHEVNGAAEF 52
Substrate F (phenylalanine) RWHHEVNFAAEF 53 mixture 2 A (alanine)
RWHHEVNAAAEF 54 V (valine) RWHHEVNVAAEF 55 L (leucine) RWHHEVNLAAEF
56 Q (glutamine) RWHHEVNQAAEF 57 M (methionine) RWHHEVNMAAEF 58 Y
(tyrosine) RWHHEVNYAAEF 59 Substrate F (phenylalanine) RWHHEVNFAAEF
60 mixture 3 P (proline) RWHHEVNPAAEF 61 N (asparagine)
RWHHEVNNAAEF 62 K (lysine) RWHHEVNKAAEF 63 R (arginine)
RWHHEVNRAAEF 64 H (histidine) RWHHEVNHAAEF 65 W (tryptophan)
RWHHEVNWAAEF 66
[0186] Initial Rate Determination by MALDI-TOF MS
[0187] Substrate mixtures were dissolved at 2 mg/ml in 10% glacial
acetic acid, and diluted into appropriate concentration of NaOH to
obtain a mixture of substrates in the .mu.M range in sodium acetate
at pH 4.1. Aliquots were equilibrated at 25 .sup.KC, and reactions
were initiated by the addition of aliquots of memapsin 2. Aliquots
(10 .mu.l) were removed at time intervals and combined with
MALDI-TOF matrix (.alpha.-hydroxycinnamic acid in acetone, 20
mg/ml) and immediately spotted in duplicate onto a stainless-steel
MALDI sample plate. Samples were subjected to analysis by using
MALDI-TOF mass spectrometry device, operated at 20,000 accelerating
volts in positive mode with a 150 nsec delay, using a PE Biosystems
Voyager DE instrument at the Molecular Biology Resource Center on
campus. Ions with a mass-to-charge ratio (m/z) were detected in the
range of 400-2000 amu (atomic mass units). Data were analyzed by
the Voyager Data Explorer module to obtain ion intensity data for
mass species of interest.
[0188] Random Sequence Inhibitor Library
[0189] The random sequence inhibitor library was based on the
sequence of OM99-2 with random amino acids (less cysteine) at 4
subsite positions P.sub.2, P.sub.3, P.sub.2' and P.sub.3'.
Diisostere Leu*Ala (* represents hydroxyethylene transition-state
isostere) was used in the synthesis to fix the positions P.sub.1
and P.sub.1'. Peptides were synthesized by solid-state peptide
synthesis method and left attached on the resin beads. By using the
`split-synthesis` procedure (Lam et al., 1991), each of the resin
beads contained only one -sequence while the sequence differed from
bead to bead. The overall library sequence was:
6 (SEQ ID NO:67) Gly-Xx1-Xx2-Leu*Ala-Xx3-Xx4-Phe-Arg-Met-Gl- y-Gly-
[Resin bead] P.sub.4 P.sub.3 P.sub.2 P.sub.1 P.sub.1' P.sub.2'
P.sub.3' P.sub.4'
[0190] where Xxn residues (where n represents either 1, 2, 3, or 4)
are randomized at each position with 19 amino acids. A shorter
version of the peptides, starting at P.sub.2' (sequence:
Xx3-Xx4-Phe-Arg-Met-Gly-Gly-[Re- sin bead] SEQ ID NO: 68), was also
present in each bead with a ratio to the longer sequence at about
7:3. Without isostere, the short sequence would not bind memapsin 2
with significant strength but its presence was convenient for
identifying the residues at by automated Edman degradation. The
residues are identified from the randomized positions as
follows:
7 Edman cycle # 1 2 3 4 Sequence 1, from the long sequence: Gly Xx1
Xx2 Leu Sequence 2, from the short sequence: Xx3 Xx4 Phe Arg
[0191] The assignment of Xx3 and Xx2 had no ambiguity since they
are the only unknown residue at cycle 1 and 3 respectively. Amino
acids Xx1 and Xx4 were assigned from their relative amounts. The
presence of a methionine would permit the CNBr cleavage follow by
peptide identified by MS/MS.
[0192] Probing of the Random Sequence Library
[0193] About 130,000 individual beads representing one copy of the
library, estimated to be contained in 1.1 ml of settled beads, was
hydrated in buffer A (50 mM Na acetate, 0.1% Triton X-100, 0.4 M
urea, 0.02% Na azide, 1 mg/ml BSA, pH 3.5; filtered with a 5 micron
filter). The beads were soaked in 3% BSA in buffer A for 1 h to
block the non-specific binding, and rinsed twice with the same
buffer. Recombinant memapsin 2 was diluted into buffer A to 4 nM
and incubated with the library for 1 h. A single stringency wash
was performed which included 6.7 .mu.M transition-state isosteric
inhibitor OM99-2 in buffer B (50 mM Na acetate, 0.1% Triton X-100,
0.02% Na azide, 1 mg/ml BSA, pH 5.5; filtered with 5 micron
filter), followed by two additional washes with buffer B containing
no OM99-2. Affinity-purified IgG specific for recombinant memapsin
2 was diluted 100 fold in buffer B and incubated 30 min with the
library. Following three washes with buffer B, affinity-purified
anti-goat-alkaline phosphatase conjugate was diluted into buffer B
(1:200) and incubated for 30 min, with three subsequent washes. A
single tablet of alkaline phosphatase substrate (BCIP/NBT) was
dissolved in 10 ml water and 1 ml applied to the beads and
incubated 1 h. Beads were resuspended in 0.02% sodium azide in
water and examined under a dissecting microscope. Darkly-stained
beads were graded by sight, individually isolated, stripped in 8 M
urea for 24 h, and destained in DMF. The sequence determination of
the beads were carried out in an Applied Biosystem Protein
Sequencer at the Molecular Biology Resource Center on campus and
the PTH-amino acids were quantified using reversed-phase HPLC.
[0194] Determination of Kinetic Parameters
[0195] The kinetic parameters, K.sub.m and k.sub.cat, using single
peptide substrate, and K.sub.i against free inhibitors, were
determined as described above.
[0196] Model Building of OM00-3 Binding to Memapsin 2
[0197] The crystal structure of OM99-2 in complex with memapsin 2
was used as the initial model. Corresponding subsite residues were
replaced with that of OM00-3. The side chain conformations were
visually selected from a rotomer library for the best fitting into
the binding pockets in terms of the non-bonded interaction. Energy
minimization was then carried out with the CNS program using Engh
and Huber energy parameters (Engh, R. A. and Huber "Accurate bond
and angle parameters for X-ray structure refinement" in: Acta
Cryst. A47, 392-400 (1991)). C.sub. atoms and the atoms with a
distance of more than 5 angstroms from the inhibitor were subject
to harmonic restrains during the minimization process.
[0198] Results
[0199] Determination of Substrate Side-Chain Preference in Memapsin
2 Sub-Sites
[0200] The substrate cleft of memapsin 2 accommodates eight
sub-sites for the side chains as shown in the crystal structure. A
complete set of side-chain preference analyzed by classical enzyme
kinetics for all sub-sites of memapsin 2 would require the
determination of 160 pairs of individual k.sub.cat and K.sub.m
values, a tedious task so far not attained for any aspartic
protease with broad specificity. The sub-site preference is,
however, defined by the relative catalytic efficiency,
k.sub.cat/K.sub.m values of substrate with different side chains,
which may be determined from the relative initial hydrolysis rates
of defined mixtures of substrates (Fersht, A. Enzyme Structure and
Mechanism, 2.sup.nd Ed., Freeman, New York, (1985), the teachings
of which are incorporated herein in their entirety). This method
has been successfully used to analyze the specificity of other
aspartic proteases (Kassel et al., 1995; Koelsch et al., 2000, the
teachings of which are incorporated herein in their entirety). The
rate determination was further simplified by the use of
MALDI-TOF/MS ion intensities for quantification of relative amounts
of products and substrates. MALDI-TOF/MS ion intensities have been
used to quantify compounds from plasma and cell culture (Sugiyama
et al, "A quantitative analysis of serum sulfatide by
matrix-assisted laser desorption ionization time-of-flight mass
spectrometry with delayed ion extraction" in: Anal. Biochem. 274:
90-97 (1999); Wu et al., "An automated MALDI mass spectrometry
approach for optimizing cyclosporin extraction and quantitation"
in: Anal Chem. 69: 3767-71 (1997), the teachings of all of which
are incorporated herein in their entirety) and pertinently, to
determine relative amount of A.beta. 40 and A.beta. 42 from cell
culture (Davies et al., "The structure and function of the aspartic
proteinases" in: Annu Rev Biophys Biophys Chem. 19: 189-215 (1990),
the teachings of which are incorporated herein in their entirety).
Advantages of MALDI-TOF/MS for this method include its sensitivity
and rapid acqusition of data. Linearity of ion intensity data with
mixtures of product and substrate produced excellent correlation,
was consistent for each substrate in the mixture, and required no
correction factor. Initial rates of hydrolysis for each peptide in
each mixture were subsequently determined using this method. Ratios
of these initial rates are proportional to their relative catalytic
efficiencies, k.sub.cat/K.sub.m (Fersht, 1985).
[0201] The relative catalytic efficiencies of memapsin 2 for eight
sub-site positions of the substrate are presented in FIG. 5. These
results confirm previous observations that none of the eight
sub-site of memapsin 2 is absolutely stringent. On both the P side
and the P' side, the central sub-sites (P.sub.1 and P.sub.1') are
more stringent than the outside sub-sites (P.sub.4 and P.sub.4').
This is in evidence when the hydrolysis efficiency of the
non-preferred residues (background) are compared to the preferred
residues. The lack of stringency is more pronounced for the 4
sub-sites on the P' side, especially for P.sub.3' and P.sub.4'
where the backgrounds are relatively high. The poor stringencies of
these two sub-sites is consistent with the observation that the
P.sub.3' and P.sub.4' side chains of OM99-2 do not significantly
interact with the enzyme in the the crystal structure.
[0202] Sub-site P.sub.4 favors Glu over Gln which, in turn, is
favored over Asp. P.sub.4-Glu of OM99-2 fits well in S4 pocket with
multiple interactions. The reduction of catalytic efficiency from
substitution of P.sub.4-Glu with Gln may be due to the loss of
charge interaction of P.sub.4 side chain with Arg.sup.235 and
Arg.sup.307. The further decrease of catalytic efficiency from
substitution of P.sub.4-Glu with Asp is likely due to the absence
of the hydrogen bond to P.sub.2-Asn. For sub-site P3, an Ile is
more preferable than Val as in OM99-2. This is due to a better
fitting in the S.sub.3 hydrophobic pocket.
[0203] Inhibitor Side Chain Preference Determined from a
Combinatorial Library
[0204] The sub-site preference for inhibitor binding was also
studied. A combinatorial library of approximately
1.3.times.10.sup.5 different inhibitors immobilized on beads was
synthesized and probed with memapsin 2. The base sequence of the
library was taken from OM99-2, EVNL*AAEF (SEQ ID NO: 69) (*
designates isostere hydroxyethylene), in which the sub-sites
P.sub.3, P.sub.2, P.sub.2', and P.sub.3' (boldface) were randomized
with all amino acids except cysteine. P.sub.1 and P.sub.1' were
keep constant due to the use of L*A in library synthesis. P4 and
P4' were not randomized in order to keep the library size
manageable. Also, the information on those outside sub-sites are
less critical for the design of smaller inhibitors. After enhancing
memapsin 2 binding selectivity to the library by washing with urea
solutions, the bound memapsin 2 on beads was detected by antibody
raised to memapsin 2 and an alkaline phophatase conjugated
secondary antibody. About 65 from about 130,000 beads were
intensely stained as observed under the light microscope. The
inhibitor sequences from the 10 most intensely stained beads were
determined by automated Edman sequencing (Table 2). A clear
consensus at the four randomized positions (boldface) was observed
to be ELDLAVEF (SEQ ID NO: 70). The clear consensus in positive
beads and the lack of consensus in negative beads (Table 2)
supports the contention that the memapsin 2 was bound to preferred
sequences in the library. Also, the consensus inhibitor sequence
agree well with the results obtained from the substrate studies. An
inhibitor, OM00-3, having the consensus sequence ELDL*AVEF (SEQ ID
NO: 71) was synthesized using the method described above. The
K.sub.i of this inhibitor proved to be 0.31 nM, nearly five fold
lower than the K.sub.i of OM99-2.
8TABLE 2 Selection of potent competitors of OM99-2 from
combinatorial library.sup.a ID P3 P2 P2' P3' SEQ ID NO. 1 Leu Asp
Val Glu 72 2 Leu Glu Val Glu 73 3 Leu Asp Val Glu 74 4 Leu Asp Val
Glu 75 5 Leu Asp Val Gln 76 6 Ile Asp Ala Gln 77 7 Ile Asp Val Tyr
78 8 Leu Glu Val Gln 79 9 Leu Phe Val Glu 80 10 Phe/Ile Ser Val
Phe/Ile 81 Neg1 Phe Met Asn Arg 82 Neg2 Asp Phe Ser 83
.sup.aLibrary template: Gly-P.sub.3-P.sub.2-Leu*Ala-P.sub.2'-P-
.sub.3'-Phe-Arg-Met-Gly-Gly-Resin (SEQ ID NOS: 72-83 )
[0205] Binding of OM00-3 in the Active Site of Memapsin 2
[0206] Comparisons between the crystal structure of OM99-2 and the
molecular model of OM2000-1 reveal improved inhibitor binding of
the later to memapsin 2. As compared to OM99-2, the larger
hydrophobic side chains at P.sub.3 and P.sub.2' subsites for OM00-3
are better accommodated by the enzyme subsite through improved van
der Waal interactions. Among these are six significant new
interactions: P.sub.3-Lew of inhibitor with Leu and Gly of the
enzyme and P.sub.2'-Val with Val, Tyr, Ile and Tyr. In addition,
the model shows that replacement of Asn by Asp at the P.sub.2
position introduces three new salt bridges between the side chain
atoms CD1 and CD2 of P.sub.2-Asp and that of NE and NH1 of Arg-235.
The inter-atomic distances of these salt bridges are 3.6, 3.4 and
3.5 angstroms respectively. These charge interactions should render
higher free energies of binding than that of P.sub.2-Asn in OM99-2.
These observed structural differences between the binding of two
inhibitors are consistent with the 4.5 fold decrease in K.sub.i
values from OM99-2 to OM00-3.
[0207] To be useful for clinical treatment of AD, memapsin 2
inhibitors must penetrate the blood-brain barrier, and
consequently, must be small in size (<700 daltons). An
eight-residue inhibitor such as OM00-3, is 917 daltons. Smaller
inhibitors can be designed using fewer sub-sites, perhaps spanning
only 4 or 5 sub-sites. Information described herein on sub-site
preferences together with the information on the three-dimensional
structure of the sub-sites can be used in the design of these
inhibitors.
EXAMPLE 6
Using The Crystal Structure to Design Inhibitors
[0208] Pharmaceutically acceptable inhibitor drugs are normally
less than 800 daltons. In the case of memapsin 2 inhibitors, this
requirement may even be more stringent due to the need for the
drugs to penetrate the blood-brain. In the current model, well
defined subsite structures at P.sub.4 to P.sub.2' provide
sufficient template areas for rational design of such drugs. The
spacial relationships of individual inhibitor side chain with the
corresponding subsite of the enzyme as revealed in this crystal
structure permits the design of new inhibitor structures in each of
these positions. It is also possible to incorporate the unique
conformation of subsites P.sub.2', P.sub.3' and P.sub.4' into the
selectivity of memapsin 2 inhibitors. The examples of inhibitor
design based on the current crystal structure are given below.
Example A
[0209] Since the side chains of P.sub.3 Val and P.sub.1 Leu are
packed against each other and there is no enzyme structure between
them, cross-linking these side chains would increase the binding
strength of inhibitor to memapsin 2. This is because when binding
to the enzyme, the cross-linked inhibitors would have less entropy
difference between the free and bound forms than their
non-cross-linked counterparts (Khan, A. R., et al., Biochemistry,
37:16839 (1998), the teachings of which are incorporated herein in
their entirety). Possible structures of the cross-linked side
chains include those shown in FIG. 1.
Example B
[0210] The same situation exits between the P4 Glu and P2 Asn. The
current crystal structure shows that these side chains are already
hydrogen bonded to each other so the cross linking between them
would also derive binding benefit as described in A. The
cross-linked structures include those shown in FIG. 2.
Example C
[0211] Based on the current crystal structure, the P1' Ala side
chain may be extended to add new hydrophobic, Van der Waals and
H-bond interactions. An example of such a design is diagramed in
FIG. 3.
Example D
[0212] Based on the current crystal structure, the polypeptide
backbone in the region of P1, P2, and P3, and the side chain of
P1-Leu can be bridged into rings by the addition of two atoms (A
and B in FIG. 4). Also, a methyl group can be added to the
beta-carbon of the P1-Leu (FIG. 4).
[0213] Modifications and variations of the methods and materials
described herein will be obvious to those skilled in the art and
are intended to come within the scope of the appended claims.
[0214] While this invention has been particularly shown and
described with references to preferred embodiments thereof, it will
be understood by those skilled in the art that various changes in
form and details may be made therein without departing from the
scope of the invention encompassed by the appended claims.
* * * * *